Cancer therapy using CLDN6 target-directed antibodies in vivo

ABSTRACT

The invention relates to the treatment and/or prevention of tumor diseases associated with cells expressing CLDN6, in particular cancer and cancer metastasis using antibodies which bind to CLDN6. The present application demonstrates that the binding of antibodies to CLDN6 on the surface of tumor cells is sufficient to inhibit growth of the tumor and to prolong survival and extend the lifespan of tumor patients. Furthermore, binding of antibodies to CLDN6 is efficient in inhibiting growth of CLDN6 positive germ cell tumors such as teratocarcinomas or embryonal carcinomas, in particular germ cell tumors of the testis.

This application is a divisional of U.S. patent application Ser. No.13/808,423, which was filed on Mar. 18, 2013 as a National Stage Entryof PCT/EP2011/003312, which was filed on Jul. 4, 2011 and claimedpriority to European Patent Application Number 10006957.4 and U.S.patent application Ser. No. 61/361,632, which were filed on Jul. 6,2010. The contents of each of the aforementioned applications areincorporated herein by reference in their entireties

Cancer is a significant health problem throughout the world and is stillamong the leading causes of death. Cancer cells biologically differsubstantially from their nonmalignant cells of origin. These differencesare due to genetic alterations acquired during cancer development andresult, inter alia, also in the formation of qualitatively orquantitatively altered molecular structures in the cancer cells.Cancer-associated structures of this kind are, in particular, geneticproducts the expression of which is induced or enhanced during thecourse of malignant transformation.

The immune system has the ability to recognize and destroy cells via twoseparate modalities: innate and adaptive immunity. The innate componentconsists of macrophages, natural killer (NK) cells, monocytes, andgranulocytes. These cells identify molecular patterns involved incellular transformation and release various cytokines and inflammatorymediators. The innate response lacks the memory capability for foreignantigens, a feature present in adaptive immune response. This lattercomponent of the immune system also features specificity for foreignantigens, imparted by the presence of receptors on lymphocytes. Antigenpresenting cells (APCs) also play a role in the adaptive response—theyengulf foreign antigens and present them to the lymphocytes in thecontext of major histocompatibility complex. CD4+ T cells bear receptorsthat recognize antigens in the context of MHC class II molecules, whichthen enables them to release cytokines and further activate CD8+lymphocytes (cytotoxic T lymphocytes; CTLs) or B cells. CTLs are part ofcell-mediated immunity and are capable of eliminating cells presented inthe context of MHC class I molecules, via apoptosis or perforin-mediatedcell lysis. It is widely accepted that T-cell mediated immunity plays avital role in an anti-tumor response. B cells are involved in release ofimmunoglobulins and as such are part of the humoral immune system.

If properly aimed and enhanced, immune functions can be therapeuticallyexploited to control and even eradicate malignant lesions. Genetic andepigenetic changes involved in carcinogenesis generate antigens that arerecognized by the immune system in analogous fashion to microbialantigens.

Antibodies have been successfully introduced into the clinic for use incancer therapy and have emerged as the most promising therapeutics inoncology over the last decade. Antibody-based therapies for cancer havethe potential of higher specificity and lower side effect profile ascompared to conventional drugs. The reason is a precise distinctionbetween normal and neoplastic cells by antibodies and the fact thattheir mode of action relies on less toxic immunological anti-tumormechanisms, such as complement activation and recruitment of cytotoxicimmune cells.

Claudins are integral membrane proteins located within the tightjunctions of epithelia and endothelia. Claudins are predicted to havefour transmembrance segments with two extracellular loops, and N- andC-termini located in the cytoplasm. The claudin (CLDN) family oftransmembrane proteins plays a critical role in the maintenance ofepithelial and endothelial tight junctions and might also play a role inthe maintenance of the cytoskeleton and in cell signaling.

We have found that CLDN6 is expressed in tissues of various cancerswhile expression in normal non-cancer tissues is limited to placenta.Such cancers include ovarian cancer, in particular ovarianadenocarcinoma and ovarian teratocarcinoma, lung cancer, including smallcell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), inparticular squamous cell lung carcinoma and adenocarcinoma, gastriccancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer,in particular basal cell carcinoma and squamous cell carcinoma,malignant melanoma, head and neck cancer, in particular malignantpleomorphic adenoma, sarcoma, in particular synovial sarcoma andcarcinosarcoma, bile duct cancer, cancer of the urinary bladder, inparticular transitional cell carcinoma and papillary carcinoma, kidneycancer, in particular renal cell carcinoma including clear cell renalcell carcinoma and papillary renal cell carcinoma, colon cancer, smallbowel cancer, including cancer of the ileum, in particular small boweladenocarcinoma and adenocarcinoma of the ileum, testicular embryonalcarcinoma, placental choriocarcinoma, cervical cancer, testicularcancer, in particular testicular seminoma, testicular teratoma andembryonic testicular cancer, and uterine cancer, and the metastaticforms thereof.

Furthermore, we were able to produce antibodies capable of specificallybinding to CLDN6 on the surface of intact cells expressing CLDN6. Nobinding to cells expressing claudin proteins other than CLDN6, inparticular, CLDN3, CLDN4 and CLDN9, or cells not expressing any of theseCLDN proteins was observed for these antibodies.

Here, we extend those observations by demonstrating that antibodybinding to CLDN6 on the surface of tumor cells is sufficient inconferring a significant tumor growth inhibition. In vivo assessment oftumor growth of tumor cells transfected with CLDN6 and non-transfectedxenografts showed the specific inhibition of tumor growth ofCLDN6-transfected cells mediated by antibody binding to CLDN6.Furthermore, it was demonstrated that antibody binding to CLDN6 issufficient in inhibiting tumor growth in vivo of endogenously CLDN6expressing tumor cells. This establishes the proof-of-principle thatantibody binding to CLDN6 is effective in inhibiting tumor growth, andprovides evidence that CLDN6 is an attractive target for therapeuticantibodies designed to inhibit tumor growth by targeting CLDN6.

Furthermore, it was demonstrated that antibody binding to CLDN6 isefficient in inhibiting tumor growth of a human CLDN6 positive germ celltumor cell line in vivo demonstrating the usefulness of antibodiesbinding to CLDN6 as selective therapeutic agents to target and inducethe killing of germ cell tumors such as testicular germ cell tumors.

Thus, we provide the first direct evidence that antibody binding toCLDN6 on the surface of tumor cells in vivo results in tumor growthattenuation and provide the demonstration that specific binding to CLDN6results in a therapeutic intervention by which tumor growth isattenuated. Furthermore, we provide evidence that antibody binding toCLDN6 on the surface of tumor cells in vivo results in the prolongationof survival and extending the lifespan of tumor patients.

Accordingly, the invention relates to the treatment and/or prevention oftumor diseases associated with cells expressing CLDN6, in particularcancer and cancer metastasis using antibodies which bind to CLDN6. Thepresent application demonstrates that the binding of antibodies to CLDN6on the surface of tumor cells is sufficient to inhibit growth of thetumor and to prolong survival and extend the lifespan of tumor patients.Furthermore, binding of antibodies to CLDN6 is efficient in inhibitinggrowth of CLDN6 positive germ cell tumors such as teratocarcinomas orembryonal carcinomas, in particular germ cell tumors of the testis.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention relates to an antibody which inhibitsgrowth of a tumor in vivo, wherein the cells of the tumor expressclaudin 6 (CLDN6) and wherein the antibody is capable of binding toCLDN6. In one embodiment, the antibody inhibits growth of the tumor bybinding to CLDN6. In one embodiment, the antibody is specific for CLDN6.In one embodiment, the antibody is a monoclonal, chimeric, human orhumanized antibody, or is a fragment of an antibody or a syntheticantibody. The tumor may be selected from the group consisting of ovariancancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma andpapillary carcinoma, kidney cancer, in particular renal cell carcinomaincluding clear cell renal cell carcinoma and papillary renal cellcarcinoma, colon cancer, small bowel cancer, including cancer of theileum, in particular small bowel adenocarcinoma and adenocarcinoma ofthe ileum, testicular embryonal carcinoma, placental choriocarcinoma,cervical cancer, testicular cancer, in particular testicular seminoma,testicular teratoma and embryonic testicular cancer, and uterine cancer,and the metastatic forms thereof. The tumor may be a germ cell tumorsuch as a teratocarcinoma or an embryonal carcinoma. The germ cell tumormay be a germ cell tumor of the testis.

In a further aspect, the invention relates to an antibody selected fromthe group consisting of (i) an antibody produced by or obtainable from aclone deposited under the accession no. DSM ACC3059 (GT512muMAB 36A),DSM ACC3058 (GT512muMAB 27A), or DSM ACC3057 (GT512muMAB 5F2D2), (ii) anantibody which is a chimerized or humanized form of the antibody under(i), (iii) an antibody which has the specificity of the antibody under(i), and (iv) an antibody comprising the antigen binding portion orantigen binding site of the antibody under (i). The antigen bindingportion or antigen binding site of the antibody under (i) may comprisethe variable region of the antibody under (i).

The antibody according to any of the above aspects may be attached to atleast one therapeutic effector moiety such as a radiolabel, cytotoxin orcytotoxic enzyme.

In a further aspect, the invention relates to a hybridoma capable ofproducing the antibody according to any of the above aspects.

In a further aspect, the invention relates to a hybridoma depositedunder the accession no. DSM ACC3059 (GT512muMAB 36A), DSM ACC3058(GT512muMAB 27A), or DSM ACC3057 (GT512muMAB 5F2D2).

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising the antibody according to any of the aboveaspects. The pharmaceutical composition may in the form of a therapeuticor prophylactic tumor vaccine. In one embodiment, the pharmaceuticalcomposition is for use in treating or preventing a tumor disease.

In a further aspect, the invention relates to a method of treating apatient having a tumor disease or being at risk of developing a tumordisease, wherein the cells of the tumor express claudin 6 (CLDN6) andwherein the method comprises the administration of an antibody capableof binding to CLDN6. In one embodiment, the antibody when administeredto the patient inhibits growth of the tumor in the patient by binding toCLDN6. In one embodiment, the antibody is attached to at least onetherapeutic effector moiety such as a radiolabel, cytotoxin or cytotoxicenzyme. The antibody may be specific for CLDN6. The antibody may be amonoclonal, chimeric, human or humanized antibody, or a fragment of anantibody or a synthetic antibody. In one embodiment the method comprisesthe administration of a pharmaceutical composition according to any ofthe above aspects.

In any of the above aspects, the tumor disease may be selected from thegroup consisting of ovarian cancer, in particular ovarian adenocarcinomaand ovarian teratocarcinoma, lung cancer, including small cell lungcancer (SCLC) and non-small cell lung cancer (NSCLC), in particularsquamous cell lung carcinoma and adenocarcinoma, gastric cancer, breastcancer, hepatic cancer, pancreatic cancer, skin cancer, in particularbasal cell carcinoma and squamous cell carcinoma, malignant melanoma,head and neck cancer, in particular malignant pleomorphic adenoma,sarcoma, in particular synovial sarcoma and carcinosarcoma, bile ductcancer, cancer of the urinary bladder, in particular transitional cellcarcinoma and papillary carcinoma, kidney cancer, in particular renalcell carcinoma including clear cell renal cell carcinoma and papillaryrenal cell carcinoma, colon cancer, small bowel cancer, including cancerof the ileum, in particular small bowel adenocarcinoma andadenocarcinoma of the ileum, testicular embryonal carcinoma, placentalchoriocarcinoma, cervical cancer, testicular cancer, in particulartesticular seminoma, testicular teratoma and embryonic testicularcancer, and uterine cancer, and the metastatic forms thereof.

In any of the above aspects, the tumor disease may be a germ cell tumordisease such as a disease characterized by a teratocarcinoma or anembryonal carcinoma. The germ cell tumor disease may be a germ celltumor disease of the testis.

In any of the above aspects, the CLDN6 may comprise an amino acidsequence encoded by a nucleic acid which comprises the nucleic acidsequence according to SEQ ID NO: 1 of the sequence listing or a variantof said nucleic acid sequence and/or may comprises the amino acidsequence according to SEQ ID NO: 2 of the sequence listing or a variantof said amino acid sequence.

An antibody described herein is capable of binding to CLDN6 and ispreferably capable of binding to CLDN6 associated with the surface of acell that expresses CLDN6. Preferably, the antibody is not substantiallycapable of binding to CLDN3, in particular when associated with thesurface of a cell that expresses CLDN3, and/or is not substantiallycapable of binding to CLDN4, in particular when associated with thesurface of a cell that expresses CLDN4. Preferably, the antibody is notsubstantially capable of binding to CLDN9, in particular when associatedwith the surface of a cell that expresses CLDN9. Most preferably, theantibody is not substantially capable of binding to a CLDN protein otherthan CLDN6, in particular when associated with the surface of a cellthat expresses said CLDN protein, and is specific for CLDN6. Preferably,said cell expressing said CLDN protein is an intact cell, in particulara non-permeabilized cell, and said CLDN protein associated with thesurface of a cell has a native, i.e. non-denatured, conformation.Preferably, the antibody is capable of binding to one or more epitopesof CLDN6 in their native conformation.

In particular preferred embodiments, an antibody described herein bindsto native epitopes of CLDN6 present on the surface of living cells suchas those of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. In furtherpreferred embodiments, the antibody is specific for CLDN6-expressingtumor cells and does not bind to tumor cells not expressing CLDN6.Preferably, an antibody described herein specifically binds to CLDN6.

In one embodiment, an antibody described herein is capable of binding toan epitope located within an extracellular portion of CLDN6, whereinsaid extracellular portion of CLDN6 preferably comprises the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, more preferablythe amino acid sequence of SEQ ID NO: 5. Preferably, the antibody iscapable of binding to an epitope located within the amino acid sequenceof SEQ ID NO: 5.

In one embodiment, the antibody is obtainable by a method comprising thestep of immunizing an animal with a peptide having the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, more preferablythe amino acid sequence of SEQ ID NO: 5 or an immunologically equivalentpeptide, or a nucleic acid or host cell expressing said peptide.

In different embodiments, the CLDN6 to which the antibody is capable ofbinding has the amino acid sequence of SEQ ID NO: 2 or the amino acidsequence of SEQ ID NO: 6. It is particularly preferred that the antibodyis capable of binding to CLDN6 having the amino acid sequence of SEQ IDNO: 2 and capable of binding to CLDN6 having the amino acid sequence ofSEQ ID NO: 6.

In preferred embodiments, an antibody described herein has one or moreof the following activities: (i) killing of a cell expressing CLDN6,(ii) inhibition of proliferation of a cell expressing CLDN6, (iii)inhibition of colony formation of a cell expressing CLDN6, and (iv)inhibition of metastasis of a cell expressing CLDN6. Killing of cells,inhibition of proliferation of cells and/or inhibition of colonyformation of cells can be utilized therapeutically for inhibiting tumorgrowth which includes stopping and/or preventing tumor growth, retardingtumor growth and/or reducing the size of an existing tumor and thus, canbe utilized therapeutically for treating or preventing cancer, cancermetastasis and/or the metastatic spread of cancer cells.

Preferably an antibody described herein mediates killing of cells byinducing complement dependent cytotoxicity (CDC) mediated lysis,antibody dependent cellular cytotoxicity (ADCC) mediated lysis,apoptosis, homotypic adhesion, and/or phagocytosis, preferably byinducing CDC mediated lysis and/or ADCC mediated lysis.

Preferably, ADCC mediated lysis of cells takes place in the presence ofeffector cells, which in particular embodiments are selected from thegroup consisting of monocytes, mononuclear cells, NK cells and PMNs, andphagocytosis is by macrophages.

The activity of inhibiting or reducing proliferation of cells expressingCLDN6, preferably cancer cells, can be measured in vitro by determiningproliferation of CLDN6-expressing cancer cells in an assay usingbromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). BrdU is a syntheticnucleoside which is an analogue of thymidine and can be incorporatedinto the newly synthesized DNA of replicating cells (during the S phaseof the cell cycle), substituting for thymidine during DNA replication.Detecting the incorporated chemical using, for example, antibodiesspecific for BrdU indicates cells that were actively replicating theirDNA.

The activity of inhibiting or reducing colony formation of cellsexpressing CLDN6, preferably cancer cells, can be measured in vitro in aclonogenic assay. A clonogenic assay is a microbiology technique forstudying the effectiveness of specific agents on the survival andproliferation of cells. It is frequently used in cancer researchlaboratories to determine the effect of drugs or radiation onproliferating tumor cells. The experiment involves three major steps:(i) applying a treatment to a sample of cells, in particular cancercells, (ii) plating the cells in a tissue culture vessel and (iii)allowing the cells to grow. The colonies produced are fixed, stained,and counted. Colony formation is of importance with respect to theformation of metastases if individual tumor cells colonize organs. Theinhibitory activity of the antibodies indicates their potential insuppressing the formation of metastases. Antibodies having the activityof inhibiting or reducing colony formation in a clonogenic assay areparticularly useful for treating or preventing metastasis and themetastatic spread of cancer cells, in particular of the cancer typesmentioned herein.

In preferred embodiments, an antibody described herein exhibits one ormore immune effector functions against a cell carrying CLDN6 in itsnative conformation, wherein the one or more immune effector functionsare preferably selected from the group consisting of complementdependent cytotoxicity (CDC), antibody-dependent cell-mediatedcytotoxicity (ADCC), induction of apoptosis, and inhibition ofproliferation, preferably the effector functions are ADCC and/or CDC.

Preferably tumor growth inhibition or immune effector functions exertedby an antibody described herein are induced by binding of said antibodyto CLDN6, preferably to an epitope located within an extracellularportion of CLDN6, wherein said extracellular portion of CLDN6 preferablycomprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 5, more preferably the amino acid sequence of SEQ ID NO: 5.

According to the invention, a cell expressing CLDN6 is preferablycharacterized by association of CLDN6 with its cell surface. A cellexpressing CLDN6 or a cell characterized by association of CLDN6 withits cell surface or carrying CLDN6 in its native conformation preferablyis a tumor cell, such as a cancer cell, preferably a cancer cell from acancer described herein.

An antibody described herein may be attached to one or more therapeuticeffector moieties, e.g., radiolabels, cytotoxins, therapeutic enzymes,agents that induce apoptosis, and the like in order to provide fortargeted cytotoxicity, i.e., killing of tumor cells.

In one embodiment an antibody described herein (i) binds to cellsexpressing CLDN6, and (ii) does not bind to cells not expressing CLDN6.An antibody described herein preferably (i) mediates killing and/orinhibits proliferation of cells expressing CLDN6, and (ii) does notmediate killing and/or does not inhibit proliferation of cells notexpressing CLDN6.

In one embodiment, an antibody described herein can be characterized byone or more of the following properties:

-   a) specificity for CLDN6;-   b) a binding affinity to CLDN6 of about 100 nM or less, preferably,    about 5-10 nM or less and, more preferably, about 1-3 nM or less;-   c) the ability to deplete tumor cells which express CLDN6;-   d) the ability to stop or retard proliferation of tumor cells which    express CLDN6;-   e) the ability to prolong survival of a subject having tumor cells    which express CLDN6.

In one embodiment, an antibody described herein reduces tumor cellgrowth and/or induces tumor cell death and thus, has a tumor-inhibitingor tumor-destroying effect.

A preferred antibody described herein is an antibody produced by orobtainable from a hybridoma cell deposited at the DSMZ (Inhoffenstr. 7B,38124 Braunschweig, Germany) and having one of the followingdesignations and accession numbers:

-   1. GT512muMAB 36A, accesssion no. DSM ACC3059, deposited on Apr. 13,    2010;-   2. GT512muMAB 27A, accesssion no. DSM ACC3058, deposited on Apr. 13,    2010; or-   3. GT512muMAB 5F2D2, accesssion no. DSM ACC3057, deposited on Apr.    13, 2010.

Antibodies of the invention are designated herein by referring to thedesignation of the antibody and/or by referring to the clone producingthe antibody, e.g. muMAB 36A.

Further preferred antibodies are those having the specificity of theantibodies produced by and obtainable from the above-describedhybridomas and, in particular, those comprising an antigen bindingportion or antigen binding site, in particular a variable region,identical or highly homologous to that of the antibodies produced by andobtainable from the above-described hybridomas. It is contemplated thatpreferred antibodies are those having CDR regions either identical orhighly homologous to the regions of antibodies produced by andobtainable from the above-described hybridomas. By “highly homologous”it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1to 3 or 1 or 2 substitutions may be made. Particularly preferredantibodies are the chimerized and humanized forms of the antibodiesproduced by and obtainable from the above-described hybridomas.

The present invention also relates to a cell such as a hybridoma cellproducing an antibody as described herein.

Preferred hybridoma cells are those deposited at the DSMZ (Inhoffenstr.7B, 38124 Braunschweig, Germany) and having one of the followingdesignations and accession numbers:

-   1. GT512muMAB 36A, accesssion no. DSM ACC3059, deposited on Apr. 13,    2010;-   2. GT512muMAB 27A, accesssion no. DSM ACC3058, deposited on Apr. 13,    2010; or-   3. GT512muMAB 5F2D2, accesssion no. DSM ACC3057, deposited on Apr.    13, 2010.

The present invention also relates to nucleic acids comprising genes ornucleic acid sequences encoding antibodies or parts thereof, e.g. anantibody chain, as described herein. The nucleic acids may be comprisedin a vector, e.g., a plasmid, cosmid, virus, bacteriophage or anothervector used e.g. conventionally in genetic engineering. The vector maycomprise further genes such as marker genes which allow for theselection of the vector in a suitable host cell and under suitableconditions. Furthermore, the vector may comprise expression controlelements allowing proper expression of the coding regions in suitablehosts. Such control elements are known to the artisan and may include apromoter, a splice cassette, and a translation initiation codon.

Preferably, the nucleic acid of the invention is operatively attached toexpression control elements allowing expression in eukaryotic orprokaryotic cells. Control elements ensuring expression in eukaryotic orprokaryotic cells are well known to those skilled in the art.

Methods for construction of nucleic acid molecules, for construction ofvectors comprising nucleic acid molecules, for introduction of vectorsinto appropriately chosen host cells, or for causing or achievingexpression of nucleic acid molecules are well-known in the art.

A further aspect of the present invention relates to a host cellcomprising a nucleic acid or vector as disclosed herein.

In one aspect, the invention provides compositions, e.g., pharmaceuticaland diagnostic compositions/kits, comprising an antibody or acombination of antibodies described herein. A pharmaceutical compositionof the invention may comprise a pharmaceutically acceptable carrier andmay optionally comprise one or more adjuvants, stabilizers etc. In aparticular embodiment, the composition includes a combination ofantibodies which bind to distinct epitopes or which possess distinctfunctional characteristics, such as inducing CDC and/or ADCC.

In one embodiment, the pharmaceutical composition of the presentinvention is a therapeutic or prophylactic anti-tumor vaccine.

In one aspect, the invention provides therapeutic and prophylacticmethods of treating a patient having a tumor disease or being at risk ofdeveloping a tumor disease. In one aspect, the invention providesmethods for inhibiting tumor growth. In one aspect, the inventionprovides methods for inducing tumor cell death. These aspects mayinvolve the administration of the antibodies or compositions describedherein to a patient.

The present invention also includes the simultaneous or sequentialadministration of two or more anti-CLDN6 antibodies, wherein preferablyat least one of said antibodies is a chimeric anti-CLDN6 antibody and atleast one further antibody is a human anti-CLDN6 antibody, theantibodies binding to the same or different epitopes of CLDN6.Preferably, a chimeric CLDN6 antibody of the invention is administeredfirst followed by the administration of a human anti-CLDN6 antibody,wherein the human anti-CLDN6 antibody is preferably administered for anextended period of time, i.e. as maintenance therapy.

An antibody or a composition described herein can be used in a varietyof methods for inhibiting growth of tumor cells expressing CLDN6 and/orselectively killing tumor cells expressing CLDN6 and thus inhibitingtumor growth by contacting the cells with an effective amount of theantibody or composition, such that the growth of the cell is inhibitedand/or the cell is killed. In one embodiment, the method includeskilling of the tumor cell expressing CLDN6, optionally in the presenceof effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis,or by a combination of two or more of these mechanisms.

An antibody or a composition described herein can be used to treatand/or prevent tumor diseases involving cells expressing CLDN6 byadministering the antibody or composition to patients suffering from orbeing at risk of developing such diseases.

The invention may involve a prophylactic and/or therapeutic treatment oftumor diseases, i.e. for treating a patient having a tumor disease orbeing at risk of developing a tumor disease. In one aspect, theinvention provides methods for inhibiting tumor growth comprising theadministration of one or more of the antibodies and compositionsdescribed herein.

Preferably, the antibodies and compositions described herein areadministered in a way such that the therapeutically active substance, inparticular the antibody, is not delivered or not substantially deliveredto a tissue or organ wherein the cells when the tissue or organ is freeof tumors express CLDN6 such as placenta tissue or placenta. To thisend, the agents and compositions described herein can be administeredlocally.

In one aspect, the invention provides an antibody as described hereinfor use in the methods of treatment described herein. In one embodiment,the invention provides a pharmaceutical composition as described hereinfor use in the methods of treatment described herein.

The treatments described herein can be combined with surgical resectionand/or radiation and/or traditional chemotherapy.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1:

Binding specificity of anti-CLDN6 murine monoclonlal antibodies muMAB5F2D2, 27A and 36A.

MuMAB 5F2D2, 27A and 36A antibodies strongly bind to cells expressingCLDN6, while they do not bind to cells expressing CLDN3 or CLDN4.

FIG. 2:

Titration of muMAB 5F2D2 binding to HEK293T cells transientlytransfected with CLDN6, 3, 4 or 9, respectively.

MuMAB 5F2D2 shows strong binding to human CLDN6 and weak binding tohuman CLDN9. The antibody does not interact with either human CLDN3 or4.

FIG. 3:

Titration of muMAB 27A binding to HEK293T cells transiently transfectedwith CLDN6, 3, 4 or 9, respectively.

MuMAB 27A shows strong binding to human CLDN6 and very weak binding tohuman CLDN9. The antibody does not interact with either human CLDN3 or4.

FIG. 4:

Titration of muMAB 36A binding to HEK293T cells transiently transfectedwith CLDN6, 3, 4 or 9, respectively.

MuMAB 36A shows strong binding to human CLDN6 and virtually no bindingto human CLDN9. The antibody does not interact with either human CLDN3or 4.

FIG. 5:

Relative affinities of anti-CLDN6 murine monoclonal antibodies muMAB5F2D2, 27A and 36A.

MuMAB 5F2D2 and 27A exhibit EC50 values of 350-450 ng/ml and saturationof binding is achieved at low concentrations whereas muMAB 36A does notshow saturation of binding even at the highest concentration.

FIG. 6:

Complement-dependent cytotoxicity (CDC) activity of anti-CLDN6 murinemonoclonal antibody muMAB 5F2D2.

MuMAB 5F2D2 shows CDC activity in a dose-dependent manner.

FIG. 7:

Complement-dependent cytotoxicity (CDC) activity of anti-CLDN6 murinemonoclonal antibodies muMAB 27A and 36A.

MuMAB 27A exhibits dose-dependent CDC activity whereas muMAB 36A is notable to induce CDC in vitro.

FIG. 8:

Induction of antibody-dependent cell-mediated cytotoxicity (ADCC) by thechimeric anti-CLDN6 antibody chimAB 5F2D2 on endogenously CLDN6expressing NEC8 and NEC8 LVTS2 54 (CLDN6 knock-down).

The chimeric anti-CLDN6 antibody chimAB 5F2D2 induces ADCC on NEC8 cellswith effector cells of two different donors in a dose dependent manner.The efficiency to induce ADCC on NEC8 LVTS2 54 cells (CLDN6 knock-down)is strongly decreased with chimAB 5F2D2.

FIG. 9:

Therapeutic effect of muMAB 5F2D2 in an early treatment xenograft model.

MuMAB 5F2D2 shows specific and strong tumor growth inhibition in miceengrafted with HEK293 cells stably expressing human CLDN6.

FIG. 10:

Therapeutic effect of muMAB 5F2D2 in an early treatment xenograft model.

Tumor volumes are significantly reduced at day 28 (and thereafter) aftertreatment with muMAB 5F2D2 in a Kruskal-Wallis test.

FIG. 11:

Therapeutic effect of muMAB 5F2D2 in an early treatment xenograft model.

Mice treated with the monoclonal murine anti-CLDN6 antibody muMAB 5F2D2show prolonged survival compared to PBS control groups.

FIG. 12:

Therapeutic effect of muMAB 27A in an early treatment xenograft model.

MuMAB 27A shows specific and strong tumor growth inhibition in miceengrafted with HEK293 cells stably expressing human CLDN6.

FIG. 13:

Therapeutic effect of muMAB 36A in an early treatment xenograft model.

MuMAB 36A shows specific and strong tumor growth inhibition in miceengrafted with HEK293 cells stably expressing human CLDN6.

FIG. 14:

Therapeutic effect of muMAB 27A and 36A in an early treatment xenograftmodel.

Mice treated with the monoclonal murine anti-CLDN6 antibodies muMAB 27Aand 36A show prolonged survival.

FIG. 15:

Immunoblot analysis of human CLDN3, 4, 6 and 9 expression in NEC8 cells.

The testicular germ cell tumor cell line NEC8 only shows expression ofCLDN6 (left panel) but not of CLDN3, 4 or 9, respectively (rightpanels).

FIG. 16:

Analysis of CLDN6 surface expression on NEC8 cells using flow cytometry.

CLDN6 is expressed on NEC8 cells.

FIG. 17:

Therapeutic effect of muMAB 5F2D2 in an early treatment xenograft modelusing mice engrafted with the tumor cell line NEC8.

Compared to the saline control group muMAB 5F2D2 showed specific andstrong tumor growth inhibition in mice engrafted with NEC8 cells thatendogenously express human CLDN6.

FIG. 18:

Therapeutic effect of muMAB 5F2D2 in an early treatment xenograft modelusing mice engrafted with the tumor cell line NEC8.

The Kruskal-Wallis test shows that tumor volumes are reduced at day 21and 42 after treatment with muMAB 5F2D2.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”), provided herein isintended merely to better illustrate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Claudins are a family of proteins that are the most important componentsof tight junctions, where they establish the paracellular barrier thatcontrols the flow of molecules in the intercellular space between cellsof an epithelium. Claudins are transmembrane proteins spanning themembrane 4 times with the N-terminal and the C-terminal end both locatedin the cytoplasm. The first extracellular loop consists on average of 53amino acids and the second one of around 24 amino acids. CLDN6 and CLDN9are the most similar members of the CLDN family.

The term “CLDN” as used herein means claudin and includes CLDN6, CLDN9,CLDN4 and CLDN3. Preferably, a CLDN is a human CLDN.

The term “CLDN6” preferably relates to human CLDN6, and, in particular,to a protein comprising (i) an amino acid sequence encoded by a nucleicacid which comprises the nucleic acid sequence according to SEQ ID NO: 1of the sequence listing or a variant of said nucleic acid sequence,and/or (ii) the amino acid sequence according to SEQ ID NO: 2 or SEQ IDNO: 6 of the sequence listing or a variant of said amino acid sequence.The first extracellular loop of CLDN6 preferably comprises amino acids28 to 80, more preferably amino acids 28 to 76 of the amino acidsequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQID NO: 6, such as the amino acid sequence shown in SEQ ID NO: 3. Thesecond extracellular loop of CLDN6 preferably comprises amino acids 138to 160, preferably amino acids 141 to 159, more preferably amino acids145 to 157 of the amino acid sequence shown in SEQ ID NO: 2 or the aminoacid sequence shown in SEQ ID NO: 6, such as the amino acid sequenceshown in SEQ ID NO: 5. Said first and/or second extracellular loopspreferably form the extracellular portion of CLDN6.

The term “CLDN9” preferably relates to human CLDN9, and, in particular,to a protein comprising the amino acid sequence according to SEQ ID NO:7 of the sequence listing or a variant of said amino acid sequence. Thefirst extracellular loop of CLDN9 preferably comprises amino acids 28 to76 of the amino acid sequence shown in SEQ ID NO: 7. The secondextracellular loop of CLDN9 preferably comprises amino acids 141 to 159of the amino acid sequence shown in SEQ ID NO: 7. Said first and/orsecond extracellular loops preferably form the extracellular portion ofCLDN9.

The term “CLDN4” preferably relates to human CLDN4, and, in particular,to a protein comprising the amino acid sequence according to SEQ ID NO:8 of the sequence listing or a variant of said amino acid sequence. Thefirst extracellular loop of CLDN4 preferably comprises amino acids 28 to76 of the amino acid sequence shown in SEQ ID NO: 8. The secondextracellular loop of CLDN4 preferably comprises amino acids 141 to 159of the amino acid sequence shown in SEQ ID NO: 8. Said first and/orsecond extracellular loops preferably form the extracellular portion ofCLDN4.

The term “CLDN3” preferably relates to human CLDN3, and, in particular,to a protein comprising the amino acid sequence according to SEQ ID NO:9 of the sequence listing or a variant of said amino acid sequence. Thefirst extracellular loop of CLDN3 preferably comprises amino acids 27 to75 of the amino acid sequence shown in SEQ ID NO: 9. The secondextracellular loop of CLDN3 preferably comprises amino acids 140 to 158of the amino acid sequence shown in SEQ ID NO: 9. Said first and/orsecond extracellular loops preferably form the extracellular portion ofCLDN3.

The above described CLDN sequences include any variants of saidsequences, in particular mutants, splice variants, conformations,isoforms, allelic variants, species variants and species homologs, inparticular those which are naturally present. An allelic variant relatesto an alteration in the normal sequence of a gene, the significance ofwhich is often unclear. Complete gene sequencing often identifiesnumerous allelic variants for a given gene. A species homolog is anucleic acid or amino acid sequence with a different species of originfrom that of a given nucleic acid or amino acid sequence. The term“CLDN” shall encompass (i) CLDN splice variants, (ii)CLDN-posttranslationally modified variants, particularly includingvariants with different glycosylation such as N-glycosylation status,(iii) CLDN conformation variants, (iv) CLDN cancer related and CLDNnon-cancer related variants. Preferably, a CLDN is present in its nativeconformation.

The term “portion” refers to a fraction. With respect to a particularstructure such as an amino acid sequence or protein the term “portion”thereof may designate a continuous or a discontinuous fraction of saidstructure. Preferably, a portion of an amino acid sequence comprises atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%,preferably at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, even more preferably at least80%, and most preferably at least 90% of the amino acids of said aminoacid sequence. Preferably, if the portion is a discontinuous fractionsaid discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or moreparts of a structure, each part being a continuous element of thestructure. For example, a discontinuous fraction of an amino acidsequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably notmore than 4 parts of said amino acid sequence, wherein each partpreferably comprises at least 5 continuous amino acids, at least 10continuous amino acids, preferably at least 20 continuous amino acids,preferably at least 30 continuous amino acids of the amino acidsequence.

The terms “part” and “fragment” are used interchangeably herein andrefer to a continuous element. For example, a part of a structure suchas an amino acid sequence or protein refers to a continuous element ofsaid structure. A portion, a part or a fragment of a structurepreferably comprises one or more functional properties of saidstructure. For example, a portion, a part or a fragment of an epitope orpeptide is preferably immunologically equivalent to the epitope orpeptide it is derived from.

The term “an extracellular portion of a CLDN” in the context of thepresent invention refers to a part of a CLDN facing the extracellularspace of a cell and preferably being accessible from the outside of saidcell, e.g., by antibodies located outside the cell. Preferably, the termrefers to one or more extracellular loops or a part thereof or any otherextracellular part of a CLDN which is preferably specific for said CLDN.Preferably, said part comprises at least 5, at least 8, at least 10, atleast 15, at least 20, at least 30, or at least 50 amino acids or more.

According to the invention, a CLDN expressed by a cell is preferablyassociated with the surface of said cell. The term “CLDN associated withthe surface of a cell” means that the CLDN is associated with andlocated at the plasma membrane of said cell, wherein at least a part ofthe CLDN, preferably the extracellular portion, faces the extracellularspace of said cell and is accessible from the outside of said cell,e.g., by antibodies located outside the cell. The association may bedirect or indirect. For example, the association may be by one or moretransmembrane domains, one or more lipid anchors, and/or by theinteraction with any other protein, lipid, saccharide, or otherstructure that can be found on the outer leaflet of the plasma membraneof a cell. For example, a CLDN associated with the surface of a cell maybe a transmembrane protein, i.e. an integral membrane protein, having anextracellular portion or may be a protein associated with the surface ofa cell by interacting with another protein that is a transmembraneprotein.

CLDN6 is associated with the surface of a cell if it is located at thesurface of said cell and is accessible to binding by CLDN6-specificantibodies added to the cell. It is to be understood that in the casewhere CLDN6 is expressed by cells, the CLDN6 associated with the surfaceof said cells may only be a portion of the expressed CLDN6.

The term “a cell carrying a CLDN” preferably means that said cellcarries a CLDN on its surface, i.e., that the CLDN is associated withthe surface of said cell.

“Cell surface” or “surface of a cell” is used in accordance with itsnormal meaning in the art, and thus includes the outside of the cellwhich is accessible to binding by proteins and other molecules.

The expression “CLDN expressed on the surface of a cell” means that theCLDN expressed by a cell is found in association with the surface ofsaid cell.

According to the invention CLDN6 is not substantially expressed in acell and is not substantially associated with a cell surface if thelevel of expression and association is lower compared to expression andassociation in placenta cells or placenta tissue. Preferably, the levelof expression and association is less than 10%, preferably less than 5%,3%, 2%, 1%, 0.5%, 0.1% or 0.05% of the expression and association inplacenta cells or placenta tissue or even lower. Preferably, CLDN6 isnot substantially expressed in a cell and is not substantiallyassociated with a cell surface if the level of expression andassociation exceeds the level of expression and association innon-tumorigenic, non-cancerous tissue other than placenta tissue by nomore than 2-fold, preferably 1,5-fold, and preferably does not exceedthe level of expression and association in said non-tumorigenic,non-cancerous tissue. Preferably, CLDN6 is not substantially expressedin a cell and is not substantially associated with a cell surface if thelevel of expression or association is below the detection limit and/orif the level of expression or association is too low to allow binding byCLDN6-specific antibodies added to the cells.

According to the invention CLDN6 is expressed in a cell and isassociated with a cell surface if the level of expression andassociation exceeds the level of expression and association innon-tumorigenic, non-cancerous tissue other than placenta tissue,preferably by more than 2-fold, preferably 10-fold, 100-fold, 1000-fold,or 10000-fold. Preferably, CLDN6 is expressed in a cell and isassociated with a cell surface if the level of expression andassociation is above the detection limit and/or if the level ofexpression and association is high enough to allow binding byCLDN6-specific antibodies added to the cells. Preferably, CLDN6expressed in a cell is expressed or exposed on the surface of said cell.

The term “raft” refers to the sphingolipid- and cholesterol-richmembrane microdomains located in the outer leaflet area of the plasmamembrane of a cell. The ability of certain proteins to associate withinsuch domains and their abbility of forming “aggregates” or “focalaggregates” can effect the protein's function. For example, thetranslocation of CLDN6 molecules into such structures, after being boundby antibodies of the present invention, creates a high density of CLDN6antigen-antibody complexes in the plasma membranes. Such a high densityof CLDN6 antigen-antibody complexes can enable efficient activation ofthe complement system during CDC.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, and includes any molecule comprising an antigen binding portionthereof. The term “antibody” includes monoclonal antibodies andfragments or derivatives thereof, including, without limitation, humanmonoclonal antibodies, humanized monoclonal antibodies, chimericmonoclonal antibodies, single chain antibodies, e.g., scFv's andantigen-binding antibody fragments such as Fab and Fab′ fragments andalso includes all recombinant forms of antibodies, e.g., antibodiesexpressed in prokaryotes, unglycosylated antibodies, and anyantigen-binding antibody fragments and derivatives as described herein.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. Each lightchain is comprised of a light chain variable region (abbreviated hereinas VL) and a light chain constant region. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system.

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another. Typically, the variable region of both light and heavychains mimics the variable regions of antibodies derived from onespecies of mammals, while the constant portions are homologous tosequences of antibodies derived from another. One clear advantage tosuch chimeric forms is that the variable region can conveniently bederived from presently known sources using readily available B-cells orhybridomas from non-human host organisms in combination with constantregions derived from, for example, human cell preparations. While thevariable region has the advantage of ease of preparation and thespecificity is not affected by the source, the constant region beinghuman, is less likely to elicit an immune response from a human subjectwhen the antibodies are injected than would the constant region from anon human source. However the definition is not limited to thisparticular example.

The term “antigen-binding portion” of an antibody (or simply “bindingportion”) refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) Fabfragments, monovalent fragments consisting of the VL, VH, CL and CHdomains; (ii) F(ab′)₂ fragments, bivalent fragments comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) Fdfragments consisting of the VH and CH domains; (iv) Fv fragmentsconsisting of the VL and VH domains of a single arm of an antibody, (v)dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consistof a VH domain; (vi) isolated complementarity determining regions (CDR),and (vii) combinations of two or more isolated CDRs which may optionallybe joined by a synthetic linker. Furthermore, although the two domainsof the Fv fragment, VL and VH, are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. A further example is binding-domainimmunoglobulin fusion proteins comprising (i) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,(ii) an immunoglobulin heavy chain CH2 constant region fused to thehinge region, and (iii) an immunoglobulin heavy chain CH3 constantregion fused to the CH2 constant region. The binding domain polypeptidecan be a heavy chain variable region or a light chain variable region.The binding-domain immunoglobulin fusion proteins are further disclosedin US 2003/0118592 and US 2003/0133939. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

The antibodies described herein are useful for passive anti-tumorimmunotherapy, and may or may not be attached to therapeutic effectormoieties, e.g., radiolabels, chemotherapeutics such as cisplatin,methotrexate, adriamycin, and the like suitable for cancer therapy,cytotoxins, therapeutic enzymes, agents that induce apoptosis, and thelike in order to provide for targeted cytotoxicity, i.e., killing oftumor cells.

Preferably the antibodies described herein mediate killing of cells byinducing complement dependent cytotoxicity (CDC) mediated lysis,antibody dependent cellular cytotoxicity (ADCC) mediated lysis,apoptosis, homotypic adhesion, and/or phagocytosis, preferably byinducing CDC mediated lysis and/or ADCC mediated lysis. The antibodiesdescribed herein preferably interact with components of the immunesystem, preferably through ADCC or CDC. However, antibodies of theinvention may also exert an effect simply by binding to tumor antigenson the cell surface, thus, e.g. blocking proliferation of the cells.

ADCC describes the cell-killing ability of effector cells as describedherein, in particular lymphocytes, which preferably requires the targetcell being marked by an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cellsand the antibody Fc domains engage Fc receptors (FcR) on the surface ofimmune effector cells. Several families of Fc receptors have beenidentified, and specific cell populations characteristically expressdefined Fc receptors. ADCC can be viewed as a mechanism to directlyinduce a variable degree of immediate tumor destruction that also leadsto antigen presentation and the induction of tumor-directed T-cellresponses. Preferably, in vivo induction of ADCC will lead totumor-directed T-cell responses and host-derived antibody responses.

CDC is another cell-killing method that can be directed by antibodies.IgM is the most effective isotype for complement activation. IgG1 andIgG3 are also both very effective at directing CDC via the classicalcomplement-activation pathway. Preferably, in this cascade, theformation of antigen-antibody complexes results in the uncloaking ofmultiple Clq binding sites in close proximity on the C_(H)2 domains ofparticipating antibody molecules such as IgG molecules (Clq is one ofthree subcomponents of complement C1). Preferably these uncloaked Clqbinding sites convert the previously low-affinity Clq-IgG interaction toone of high avidity, which triggers a cascade of events involving aseries of other complement proteins and leads to the proteolytic releaseof the effector-cell chemotactic/activating agents C3a and C5a.Preferably, the complement cascade ends in the formation of a membraneattack complex, which creates pores in the cell membrane that facilitatefree passage of water and solutes into and out of the cell and may leadto apoptosis.

The term “antibody” includes “bispecific molecules”, i.e. moleculeswhich have two different binding specificities. For example, themolecule may bind to, or interact with (a) a cell surface antigen, and(b) an Fc receptor on the surface of an effector cell. The term“antibody” also includes “multispecific molecules” or “heterospecificmolecules”, i.e. molecules which have more than two different bindingspecificities. For example, the molecule may bind to, or interact with(a) a cell surface antigen, (b) an Fc receptor on the surface of aneffector cell, and (c) at least one other component. Accordingly, theinvention includes, but is not limited to, bispecific, trispecific,tetraspecific, and other multispecific molecules which are directed toCLDN6, and to other targets, such as Fc receptors on effector cells. Theterm “antibody” also includes “bispecific antibodies” which also includediabodies. Diabodies are bivalent, bispecific antibodies in which the VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

The term “antibody” also includes “heteroantibodies” which term refersto two or more antibodies, derivatives thereof, or antigen bindingregions linked together, at least two of which have differentspecificities. These different specificities include a bindingspecificity for an Fc receptor on an effector cell, and a bindingspecificity for an antigen or epitope on a target cell, e.g., a tumorcell.

“Target cell” shall mean any undesirable cell in a subject (e.g., ahuman or animal) that can be targeted by an antibody of the invention.In preferred embodiments, the target cell is a cell expressing CLDN6.Cells expressing CLDN6 typically include tumor cells.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include cells of myeloid or lymphoid origin, e.g,lymphocytes (e.g., B cells and T cells including cytolytic T cells(cytotoxic T lymphocytes; CTLs), killer cells, natural killer cells,macrophages, monocytes, eosinophils, neutrophils, polymorphonuclearcells, granulocytes, mast cells, and basophils. Some effector cellsexpress specific Fc receptors and carry out specific immune functions.In preferred embodiments, an effector cell is capable of inducingantibody-dependent cellular cytotoxicity (ADCC), e.g., a neutrophilcapable of inducing ADCC. For example, monocytes, macrophages, whichexpress FcR are involved in specific killing of target cells andpresenting antigens to other components of the immune system, or bindingto cells that present antigens. In other embodiments, an effector cellcan phagocytose a target antigen, target cell, or microorganism. Theexpression of a particular FcR on an effector cell can be regulated byhumoral factors such as cytokines. For example, expression of Fc-gammaRIhas been found to be up-regulated by interferon gamma (IFN-γ). Thisenhanced expression increases the cytotoxic activity ofFc-gammaRI-bearing cells against targets. An effector cell canphagocytose or lyse a target antigen or a target cell.

The antibodies described herein may be human antibodies. The term “humanantibody”, as used herein, is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo).

The antibodies described herein may be monoclonal antibodies. The term“monoclonal antibody” as used herein refers to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodydisplays a single binding specificity and affinity for a particularepitope. In one embodiment, the monoclonal antibodies are produced by ahybridoma which includes a B cell obtained from a non-human animal,e.g., mouse, fused to an immortalized cell.

The antibodies described herein may be recombinant antibodies. The term“recombinant antibody”, as used herein, includes all antibodies that areprepared, expressed, created or isolated by recombinant means, such as(a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a “heterologous antibody” is defined in relation to atransgenic organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic organism, and being generally derived from a speciesother than the transgenic organism.

As used herein, a “heterohybrid antibody” refers to an antibody havinglight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

The invention includes all antibodies and derivatives of antibodies asdescribed herein which for the purposes of the invention are encompassedby the term “antibody”. The term “antibody derivatives” refers to anymodified form of an antibody, e.g., a conjugate of the antibody andanother agent or antibody, or an antibody fragment.

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCLDN6 is substantially free of antibodies that specifically bindantigens other than CLDN6). An isolated antibody that specifically bindsto an epitope, isoform or variant of human CLDN6 may, however, havecross-reactivity to other related antigens, e.g., from other species(e.g., CLDN6 species homologs). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies relates to antibodies having different specificities andbeing combined in a well defined composition.

According to the present invention, an antibody is capable of binding toa predetermined target if it has a significant affinity for saidpredetermined target and binds to said predetermined target in standardassays. “Affinity” or “binding affinity” is often measured byequilibrium dissociation constant (K_(D)). Preferably, the term“significant affinity” refers to the binding to a predetermined targetwith a dissociation constant (K_(D)) of 10⁻⁵ M or lower, 10⁻⁶ M orlower, 10⁻⁷ M or lower, 10⁻⁸M or lower, 10⁻⁹M or lower, 10⁻¹⁰M or lower,10⁻¹¹M or lower, or 10⁻¹²M or lower.

An antibody is not (substantially) capable of binding to a target if ithas no significant affinity for said target and does not bindsignificantly to said target in standard assays. Preferably, an antibodyis not (substantially) capable of binding to a target if it does notdetectably bind to said target in a flow cytometry analysis (FACSanalysis) wherein binding of said antibody to said target expressed onthe surface of intact cells is determined. Preferably, the antibody doesnot detectably bind to said target if present in a concentration of upto 2, preferably 10, more preferably 20, in particular 50 or 100 μg/mlor higher. Preferably, an antibody has no significant affinity for atarget if it binds to said target with a K_(D) that is at least 10-fold,100-fold, 10³-fold, 10⁴-fold, 10⁵-fold, or 10⁶-fold higher than theK_(D) for binding to the predetermined target to which the antibody iscapable of binding. For example, if the K_(D) for binding of an antibodyto the target to which the antibody is capable of binding is 10⁻⁷ M, theK_(D) for binding to a target for which the antibody has no significantaffinity would be is at least 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or10⁻¹ M.

An antibody according to the present invention is preferably capable ofbinding specifically to a predetermined target, in particular CLDN6.

An antibody is specific for a predetermined target if it is capable ofbinding to said predetermined target while it is not capable of bindingto other targets, i.e. has no significant affinity for other targets anddoes not significantly bind to other targets in standard assays.According to the invention, an antibody is specific for CLDN6 if it iscapable of binding to CLDN6 but is not (substantially) capable ofbinding to other targets, in particular claudin proteins other thanCLDN6 such as CLDN9, CLDN4, CLDN3 and CLDN1. Preferably, an antibody isspecific for CLDN6 if the affinity for and the binding to a claudinprotein other than CLDN6 such as CLDN9, CLDN4, CLDN3 and CLDN1 does notsignificantly exceed the affinity for or binding to claudin-unrelatedproteins such as bovine serum albumin (BSA), casein, human serum albumin(HSA) or non-claudin transmembrane proteins such as MHC molecules ortransferrin receptor or any other specified polypeptide. Preferably, anantibody is specific for a predetermined target if it binds to saidtarget with a K_(D) that is at least 10-fold, 100-fold, 10³-fold,10⁴-fold, 10⁵-fold, or 10⁶-fold lower than the K_(D) for binding to atarget for which it is not specific. For example, if the K_(D) forbinding of an antibody to the target for which it is specific is 10⁻⁷ M,the K_(D) for binding to a target for which it is not specific would beat least 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

Binding of an antibody to a target can be determined experimentallyusing any suitable method; see, for example, Berzofsky et al.,“Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press New York, N.Y. (1984), Kuby, Janis Immunology, W. H.Freeman and Company New York, N.Y. (1992), and methods described herein.Affinities may be readily determined using conventional techniques, suchas by equilibrium dialysis; by using the BIAcore 2000 instrument, usinggeneral procedures outlined by the manufacturer; by radioimmunoassayusing radiolabeled target antigen; or by another method known to theskilled artisan. The affinity data may be analyzed, for example, by themethod of Scatchard et al., Ann N.Y. Acad. Sci., 51:660 (1949). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions, e.g., salt concentration, pH.Thus, measurements of affinity and other antigen-binding parameters,e.g., K_(D), IC₅₀, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. Antibodiesaccording to the invention include polyclonal and monoclonal antibodiesand include IgG2a (e.g. IgG2a, κ, λ), IgG2b (e.g. IgG2b, κ, λ), IgG3(e.g. IgG3, κ, λ) and IgM antibodies. However, other antibody isotypesare also encompassed by the invention, including IgG1, IgA1, IgA2,secretory IgA, IgD, and IgE antibodies.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin (antibody) gene locus can be identified bycomparison to germline DNA; a rearranged locus will have at least onerecombined heptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

According to the invention, antibodies may be derived from differentspecies, including but not limited to mouse, rat, rabbit, guinea pig andhuman. Antibodies also include chimeric molecules in which an antibodyconstant region derived from one species, preferably human, is combinedwith the antigen binding site derived from another species. Moreover,antibodies include humanized molecules in which the antigen bindingsites of an antibody derived from a non-human species are combined withconstant and framework regions of human origin.

Antibodies can be produced by a variety of techniques, includingconventional monoclonal antibody methodology, e.g., the standard somaticcell hybridization technique of Kohler and Milstein, Nature 256: 495(1975). Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibodies can beemployed, e.g., viral or oncogenic transformation of B-lymphocytes orphage display techniques using libraries of antibody genes.

The preferred animal system for preparing hybridomas that secretemonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Other preferred animal systems for preparing hybridomas that secretemonoclonal antibodies are the rat and the rabbit system (e.g. describedin Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995),see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodiesdirected against CLDN6 can be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice known as HuMAb mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice.” The production ofhuman antibodies in such transgenic mice can be performed as describedin detail for CD20 in WO2004 035607

Yet another strategy for generating monoclonal antibodies is to directlyisolate genes encoding antibodies from lymphocytes producing antibodiesof defined strategy e.g. see Babcock et al., 1996; A novel strategy forgenerating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined strategy. For details of recombinantantibody engineering see also Welschof and Kraus, Recombinant antibodesfor cancer therapy ISBN-0-89603-918-8 and Benny K. C. Lo AntibodyEngineering ISBN 1-58829-092-1.

To generate antibodies to CLDN6, mice can be immunized withcarrier-conjugated peptides derived from the CLDN6 sequence, an enrichedpreparation of recombinantly expressed CLDN6 antigen or fragmentsthereof and/or cells expressing CLDN6 or fragments thereof, asdescribed. Alternatively, mice can be immunized with DNA encoding fulllength human CLDN6 or fragments thereof. In the event that immunizationsusing a purified or enriched preparation of the CLDN6 antigen do notresult in antibodies, mice can also be immunized with cells expressingCLDN6, e.g., a cell line, to promote immune responses.

The immune response can be monitored over the course of the immunizationprotocol with plasma and serum samples being obtained by tail vein orretroorbital bleeds. Mice with sufficient titers of anti-CLDN6immunoglobulin can be used for fusions. Mice can be boostedintraperitonealy or intravenously with CLDN6 expressing cells 3-5 daysbefore sacrifice and removal of the spleen to increase the rate ofspecific antibody secreting hybridomas.

To generate hybridomas producing monoclonal antibodies to CLDN6, cellsfrom lymph nodes, spleens or bone marrow obtained from immunized micecan be isolated and fused to an appropriate immortalized cell line, suchas a mouse myeloma cell line. The resulting hybridomas can then bescreened for the production of antigen-specific antibodies. Individualwells can then be screened by ELISA for antibody secreting hybridomas.By Immunofluorescence and FACS analysis using CLDN6 expressing cells,antibodies with specificity for CLDN6 can be identified. The antibodysecreting hybridomas can be replated, screened again, and if stillpositive for anti-CLDN6 monoclonal antibodies can be subcloned bylimiting dilution. The stable subclones can then be cultured in vitro togenerate antibody in tissue culture medium for characterization.

Antibodies of the invention can also be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as are well known in the art(Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibodygenes, can be ligated into an expression vector such as a eukaryoticexpression plasmid such as used by the GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such as CHOcells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively othereukaryotic cells like plant derived cells, fungal or yeast cells. Themethod used to introduce these genes can be methods described in the artsuch as electroporation, lipofectine, lipofectamine or others. Afterintroduction of these antibody genes in the host cells, cells expressingthe antibody can be identified and selected. These cells represent thetransfectomas which can then be amplified for their expression level andupscaled to produce antibodies. Recombinant antibodies can be isolatedand purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,e.g. E. coli. Furthermore, the antibodies can be produced in transgenicnon-human animals, such as in milk from sheep and rabbits or in eggsfrom hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J.Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth.231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Murine monoclonal antibodies can be used as therapeutic antibodies inhumans when labeled with toxins or radioactive isotopes. Nonlabeledmurine antibodies are highly immunogenic in man when repetitivelyapplied leading to reduction of the therapeutic effect. The mainimmunogenicity is mediated by the heavy chain constant regions. Theimmunogenicity of murine antibodies in man can be reduced or completelyavoided if respective antibodies are chimerized or humanized. Chimericantibodies are antibodies, the different portions of which are derivedfrom different animal species, such as those having a variable regionderived from a murine antibody and a human immunoglobulin constantregion. Chimerisation of antibodies is achieved by joining of thevariable regions of the murine antibody heavy and light chain with theconstant region of human heavy and light chain (e.g. as described byKraus et al., in Methods in Molecular Biology series, Recombinantantibodies for cancer therapy ISBN-0-89603-918-8). In a preferredembodiment, chimeric antibodies are generated by joining humankappa-light chain constant region to murine light chain variable region.In an also preferred embodiment, chimeric antibodies can be generated byjoining human lambda-light chain constant region to murine light chainvariable region. The preferred heavy chain constant regions forgeneration of chimeric antibodies are IgG1, IgG3 and IgG4. Otherpreferred heavy chain constant regions for generation of chimericantibodies are IgG2, IgA, IgD and IgM.

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A 86: 10029-10033). Such frameworksequences can be obtained from public DNA databases that includegermline antibody gene sequences. These germline sequences will differfrom mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V (D) J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual positions evenly across the variable region. For example,somatic mutations are relatively infrequent in the amino terminalportion of framework region 1 and in the carboxy-terminal portion offramework region 4. Furthermore, many somatic mutations do notsignificantly alter the binding properties of the antibody. For thisreason, it is not necessary to obtain the entire DNA sequence of aparticular antibody in order to recreate an intact recombinant antibodyhaving binding properties similar to those of the original antibody (seeWO 99/45962). Partial heavy and light chain sequences spanning the CDRregions are typically sufficient for this purpose. The partial sequenceis used to determine which germline variable and joining gene segmentscontributed to the recombined antibody variable genes. The germlinesequence is then used to fill in missing portions of the variableregions. Heavy and light chain leader sequences are cleaved duringprotein maturation and do not contribute to the properties of the finalantibody. To add missing sequences, cloned cDNA sequences can becombined with synthetic oligonucleotides by ligation or PCRamplification. Alternatively, the entire variable region can besynthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has certain advantages such as elimination orinclusion or particular restriction sites, or optimization of particularcodons.

The nucleotide sequences of heavy and light chain transcripts fromhybridomas are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266: 19867-19870); and HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotides approximately at the midpoint of thecorresponding non-coding oligonucleotide. Thus, for each chain, theoligonucleotides can be assembled into overlapping double stranded setsthat span segments of 150-400 nucleotides. The pools are then used astemplates to produce PCR amplification products of 150-400 nucleotides.Typically, a single variable region oligonucleotide set will be brokendown into two pools which are separately amplified to generate twooverlapping PCR products. These overlapping products are then combinedby PCR amplification to form the complete variable region. It may alsobe desirable to include an overlapping fragment of the heavy or lightchain constant region in the PCR amplification to generate fragmentsthat can easily be cloned into the expression vector constructs.

The reconstructed chimerized or humanized heavy and light chain variableregions are then combined with cloned promoter, leader, translationinitiation, constant region, 3′ untranslated, polyadenylation, andtranscription termination sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains. Plasmids for use in construction ofexpression vectors for human IgGκ are described. The plasmids can beconstructed so that PCR amplified V heavy and V kappa light chain cDNAsequences can be used to reconstruct complete heavy and light chainminigenes. These plasmids can be used to express completely human, orchimeric IgG1, Kappa or IgG4, Kappa antibodies. Similar plasmids can beconstructed for expression of other heavy chain isotypes, or forexpression of antibodies comprising lambda light chains.

Thus, in another aspect of the invention, the structural features of theanti-CLDN6 antibodies described herein, are used to create structurallyrelated humanized anti-CLDN6 antibodies that retain at least onefunctional property of the antibodies of the invention, such as bindingto CLDN6. More specifically, one or more CDR regions of mouse monoclonalantibodies can be combined recombinantly with known human frameworkregions and CDRs to create additional, recombinantly-engineered,humanized anti-CLDN6 antibodies.

The ability of an antibody to bind CLDN6 can be determined usingstandard binding assays, such as those set forth in the examples (e.g.,ELISA, Western Blot, Immunofluorescence and flow cytometric analysis)

The term “epitope” refers to an antigenic determinant in a molecule,i.e., to the part in a molecule that is recognized by the immune system,for example, that is recognized by an antibody. For example, epitopesare the discrete, three-dimensional sites on an antigen, which arerecognized by the immune system. In the context of the presentinvention, the epitope is preferably derived from a CLDN protein.Epitopes usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. An epitope of aprotein such as a CLDN preferably comprises a continuous ordiscontinuous portion of said protein and is preferably between 5 and100, preferably between 5 and 50, more preferably between 8 and 30, mostpreferably between 10 and 25 amino acids in length, for example, theepitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 amino acids in length.

The term “discontinuous epitope” as used herein, means a conformationalepitope on a protein antigen which is formed from at least two separateregions in the primary sequence of the protein.

According to the invention, the term “binding” preferably relates to aspecific binding. “Specific binding” means that an agent such as anantibody binds stronger to a target such as an epitope for which it isspecific compared to the binding to another target. An agent bindsstronger to a first target compared to a second target if it binds tothe first target with a dissociation constant (K_(D)) which is lowerthan the dissociation constant for the second target. Preferably thedissociation constant (K_(D)) for the target to which the agent bindsspecifically is more than 10²-fold, 10³-fold, 10⁴-fold, 10⁵-fold,10⁶-fold, 10⁷-fold, 10⁸-fold, 10⁹-fold, or 10¹⁰-fold lower than thedissociation constant (K_(D)) for the target to which the agent does notbind specifically.

The term “nucleic acid”, as used herein, is intended to includedeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acidscomprise according to the invention genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the invention, a nucleic acid may be present as a single-stranded ordouble-stranded and linear or covalently circularly closed molecule.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

Nucleic acids may, according to the invention, be present alone or incombination with other nucleic acids, which may be homologous orheterologous. In preferred embodiments, a nucleic acid is functionallylinked to expression control sequences which may be homologous orheterologous with respect to said nucleic acid wherein the term“homologous” means that the nucleic acid is also functionally linked tothe expression control sequence naturally and the term “heterologous”means that the nucleic acid is not functionally linked to the expressioncontrol sequence naturally.

A nucleic acid, such as a nucleic acid expressing RNA and/or protein orpeptide, and an expression control sequence are “functionally” linked toone another, if they are covalently linked to one another in such a waythat expression or transcription of said nucleic acid is under thecontrol or under the influence of said expression control sequence. Ifthe nucleic acid is to be translated into a functional protein, then,with an expression control sequence functionally linked to a codingsequence, induction of said expression control sequence results intranscription of said nucleic acid, without causing a frame shift in thecoding sequence or said coding sequence not being capable of beingtranslated into the desired protein or peptide.

The term “expression control sequence” or “expression control element”comprises according to the invention promoters, ribosome binding sites,enhancers and other control elements which regulate transcription of agene or translation of a mRNA. In particular embodiments of theinvention, the expression control sequences can be regulated. The exactstructure of expression control sequences may vary as a function of thespecies or cell type, but generally comprises 5′-untranscribed and 5′-and 3′-untranslated sequences which are involved in initiation oftranscription and translation, respectively, such as TATA box, cappingsequence, CAAT sequence, and the like. More specifically,5′-untranscribed expression control sequences comprise a promoter regionwhich includes a promoter sequence for transcriptional control of thefunctionally linked nucleic acid. Expression control sequences may alsocomprise enhancer sequences or upstream activator sequences.

According to the invention the term “promoter” or “promoter region”relates to a nucleic acid sequence which is located upstream (5′) to thenucleic acid sequence being expressed and controls expression of thesequence by providing a recognition and binding site for RNA-polymerase.The “promoter region” may include further recognition and binding sitesfor further factors which are involved in the regulation oftranscription of a gene. A promoter may control the transcription of aprokaryotic or eukaryotic gene. Furthermore, a promoter may be“inducible” and may initiate transcription in response to an inducingagent or may be “constitutive” if transcription is not controlled by aninducing agent. A gene which is under the control of an induciblepromoter is not expressed or only expressed to a small extent if aninducing agent is absent. In the presence of the inducing agent the geneis switched on or the level of transcription is increased. This ismediated, in general, by binding of a specific transcription factor.

Promoters which are preferred according to the invention includepromoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMVpromoter, and artificial hybrid promoters thereof (e.g. CMV) where apart or parts are fused to a part or parts of promoters of genes ofother cellular proteins such as e.g. human GAPDH(glyceraldehyde-3-phosphate dehydrogenase), and including or notincluding (an) additional intron(s).

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein/peptide. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.According to the invention, the term expression also includes an“aberrant expression” or “abnormal expression”.

“Aberrant expression” or “abnormal expression” means according to theinvention that expression is altered, preferably increased, compared toa reference, preferably compared to the state in a non-tumorigenicnormal cell or a healthy individual. An increase in expression refers toan increase by at least 10%, in particular at least 20%, at least 50%,at least 100%, at least 200%, at least 500%, at least 1000%, at least10000% or even more. In one embodiment, expression is only found in adiseased tissue, while expression in a healthy tissue is repressed.

In a preferred embodiment, a nucleic acid molecule is according to theinvention present in a vector, where appropriate with a promoter, whichcontrols expression of the nucleic acid. The term “vector” is used herein its most general meaning and comprises any intermediary vehicle for anucleic acid which enables said nucleic acid, for example, to beintroduced into prokaryotic and/or eukaryotic cells and, whereappropriate, to be integrated into a genome. Vectors of this kind arepreferably replicated and/or expressed in the cells. Vectors compriseplasmids, phagemids, bacteriophages or viral genomes. The term “plasmid”as used herein generally relates to a construct of extrachromosomalgenetic material, usually a circular DNA duplex, which can replicateindependently of chromosomal DNA.

As the vector for expression of an antibody, either of a vector type inwhich the antibody heavy chain and light chain are present in differentvectors or a vector type in which the heavy chain and light chain arepresent in the same vector can be used.

The teaching given herein with respect to specific nucleic acid andamino acid sequences, e.g. those shown in the sequence listing, is to beconstrued so as to also relate to modifications of said specificsequences resulting in sequences which are functionally equivalent tosaid specific sequences, e.g. amino acid sequences exhibiting propertiesidentical or similar to those of the specific amino acid sequences andnucleic acid sequences encoding amino acid sequences exhibitingproperties identical or similar to those of the amino acid sequencesencoded by the specific nucleic acid sequences.

Similarly, the teaching given herein with respect to specific antibodiesor hybridomas producing specific antibodies is to be construed so as toalso relate to antibodies characterized by an amino acid sequence and/ornucleic acid sequence which is modified compared to the amino acidsequence and/or nucleic acid sequence of the specific antibodies butbeing functionally equivalent. One important property is to retainbinding of an antibody to its target or to sustain effector functions ofan antibody. Preferably, a sequence modified with respect to a specificsequence, when it replaces the specific sequence in an antibody retainsbinding of said antibody to the target and preferably functions of saidantibody as described herein, e.g. CDC mediated lysis or ADCC mediatedlysis.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR, hypervariable and variable regions can bemodified without losing the ability to bind to a target. For example,CDR regions will be either identical or highly homologous to the regionsof antibodies specified herein. By “highly homologous” it iscontemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or1 or 2 substitutions may be made in the CDRs. In addition, thehypervariable and variable regions may be modified so that they showsubstantial homology with the regions of antibodies specificallydisclosed herein.

It is to be understood that the specific nucleic acids described hereinalso include nucleic acids modified for the sake of optimizing the codonusage in a particular host cell or organism. Differences in codon usageamong organisms can lead to a variety of problems concerningheterologous gene expression. Codon optimization by changing one or morenucleotides of the original sequence can result in an optimization ofthe expression of a nucleic acid, in particular in optimization oftranslation efficacy, in a homologous or heterologous host in which saidnucleic acid is to be expressed. For example, if nucleic acids derivedfrom human and encoding constant regions and/or framework regions ofantibodies are to be used according to the present invention, e.g. forpreparing chimeric or humanized antibodies, it may be preferred tomodify said nucleic acids for the sake of optimization of codon usage,in particular if said nucleic acids, optionally fused to heterologousnucleic acids such as nucleic acids derived from other organisms asdescribed herein, are to be expressed in cells from an organismdifferent from human such as mouse or hamster. For example, the nucleicacid sequences encoding human light and heavy chain constant regions canbe modified to include one or more, preferably, at least 1, 2, 3, 4, 5,10, 15, 20 and preferably up to 10, 15, 20, 25, 30, 50, 70 or 100 ormore nucleotide replacements resulting in an optimized codon usage. Suchnucleotide replacements preferably relate to replacements of nucleotidesnot resulting in a change in the encoded amino acid sequence or relateto corresponding replacements at corresponding positions in othernucleic acid sequences encoding human light and heavy chain constantregions, respectively.

Preferably the degree of identity between a specific nucleic acidsequence and a nucleic acid sequence which is modified with respect toor which is a variant of said specific nucleic acid sequence will be atleast 70%, preferably at least 75%, more preferably at least 80%, evenmore preferably at least 90% or most preferably at least 95%, 96%, 97%,98% or 99%. Regarding CLDN6 nucleic acid variants, the degree ofidentity is preferably given for a region of at least about 300, atleast about 400, at least about 450, at least about 500, at least about550, at least about 600 or at least about 630 nucleotides. In preferredembodiments, the degree of identity is given for the entire length ofthe reference nucleic acid sequence, such as the nucleic acid sequencesgiven in the sequence listing. Preferably, the two sequences are capableof hybridizing and forming a stable duplex with one another, withhybridization preferably being carried out under conditions which allowspecific hybridization between polynucleotides (stringent conditions).Stringent conditions are described, for example, in Molecular Cloning: ALaboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold SpringHarbor Laboratory press, Cold Spring Harbor, N.Y., 1989 or CurrentProtocols in Molecular Biology, F. M. Ausubel et al., Editors, JohnWiley & Sons, Inc., New York and refer, for example, to hybridization at65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH 7),0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodiumcitrate, pH 7. After hybridization, the membrane to which the DNA hasbeen transferred is washed, for example, in 2×SSC at room temperatureand then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68° C.

The term “variant” with respect to, for example, nucleic acid and aminoacid sequences, according to the invention includes any variants, inparticular mutants, splice variants, conformations, isoforms, allelicvariants, species variants and species homologs, in particular thosewhich are naturally present. An allelic variant relates to an alterationin the normal sequence of a gene, the significance of which is oftenunclear. Complete gene sequencing often identifies numerous allelicvariants for a given gene. A species homolog is a nucleic acid or aminoacid sequence with a different species of origin from that of a givennucleic acid or amino acid sequence.

With respect to nucleic acid molecules, the term “variant” includesdegenerate nucleic acid sequences, wherein a degenerate nucleic acidaccording to the invention is a nucleic acid that differs from areference nucleic acid in codon sequence due to the degeneracy of thegenetic code.

Furthermore, a “variant” of a specific nucleic acid sequence accordingto the invention includes nucleic acid sequences comprising single ormultiple such as at least 2, at least 4, or at least 6 and preferably upto 3, up to 4, up to 5, up to 6, up to 10, up to 15, or up to 20nucleotide substitutions, deletions and/or additions.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid deletionvariants and/or amino acid substitution variants.

In the case of amino acid sequence variants having an insertion, one ormore amino acid residues are inserted into a particular site in an aminoacid sequence, although random insertion with appropriate screening ofthe resulting product is also possible.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties.

Preferably, amino acid changes in protein variants are conservativeamino acid changes, i.e., substitutions of similarly charged oruncharged amino acids.

Preferably the degree of similarity, preferably identity between aspecific amino acid sequence and an amino acid sequence which ismodified with respect to or which is a variant of said specific aminoacid sequence such as between amino acid sequences showing substantialhomology will be at least 70%, preferably at least 80%, even morepreferably at least 90% or most preferably at least 95%, 96%, 97%, 98%or 99%. Regarding CLDN6 polypeptide variants, the degree of similarityor identity is given preferably for a region of at least about 100, atleast about 120, at least about 140, at least about 160, at least about180, at least about 200, at least about 210 amino acids. In preferredembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence such as the aminoacid sequences given in the sequence listing.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two polypeptide or nucleicacid sequences indicates the percentage of amino acids or nucleotidesthat are identical between the sequences.

The term “percentage identity” is intended to denote a percentage ofnucleotides or of amino acid residues which are identical between thetwo sequences to be compared, obtained after the best alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly and over their entire length.Sequence comparisons between two nucleotide or amino acid sequences areconventionally carried out by comparing these sequences after havingaligned them optimally, said comparison being carried out by segment orby “window of comparison” in order to identify and compare local regionsof sequence similarity. The optimal alignment of the sequences forcomparison may be produced, besides manually, by means of the localhomology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482,by means of the local homology algorithm of Neddleman and Wunsch, 1970,J. Mol. Biol. 48, 443, by means of the similarity search method ofPearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or bymeans of computer programs which use these algorithms (GAP, BESTFIT,FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

“Conservative substitutions,” may be made, for instance, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example: (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Some preferredsubstitutions may be made among the following groups: (i) S and T; (ii)P and G; and (iii) A, V, L and I. Given the known genetic code, andrecombinant and synthetic DNA techniques, the skilled scientist readilycan construct DNAs encoding the conservative amino acid variants.

The invention includes derivatives of the nucleic acid sequences, aminoacid sequences, peptides or proteins, in particular antibodies,described herein.

The term “derivative” comprises any chemical derivatization of a nucleicacid on a nucleotide base, on the sugar or on the phosphate. The term“derivative” also comprises nucleic acids which contain nucleotides andnucleotide analogs not occurring naturally. Preferably, a derivatizationof a nucleic acid increases its stability.

According to the invention, “derivatives” of proteins and peptides aremodified forms of proteins and peptides. Such modifications include anychemical modification and comprise single or multiple substitutions,deletions and/or additions of any molecules associated with the proteinor peptide, such as carbohydrates, lipids and/or proteins or peptides.The term “derivative” also extends to all functional chemicalequivalents of said proteins and peptides. Preferably, a modifiedpeptide has increased stability and/or increased immunogenicity.

According to the invention, a variant, derivative, modified form,fragment, part or portion of a nucleic acid sequence, amino acidsequence, peptide or protein preferably has a functional property of thenucleic acid sequence, amino acid sequence, peptide or protein,respectively, from which it has been derived. Such functional propertiescomprise the interaction with peptides or proteins such as antibodies orantibody targets, in particular CLDN6, the selective binding of nucleicacids and an enzymatic activity. In one embodiment, a variant,derivative, modified form, fragment, part or portion of a nucleic acidsequence, amino acid sequence, peptide or protein is immunologicallyequivalent to the nucleic acid sequence, amino acid sequence, peptide orprotein, respectively, from which it has been derived. In oneembodiment, the functional property is an immunological property.

According to the invention a cell preferably is an intact cell, i.e. acell with an intact membrane that has not released its normalintracellular components such as enzymes, organelles, or geneticmaterial. An intact cell preferably is a viable cell, i.e. a living cellcapable of carrying out its normal metabolic functions. Preferably, acell is a human cell.

A “cell” may be a “host cell” which, as used herein, is intended torefer to a cell into which a recombinant nucleic acid has beenintroduced.

The terms “transgenic animal” refers to an animal having a genomecomprising one or more transgenes, preferably heavy and/or light chaintransgenes, or transchromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is preferably capableof expressing the transgenes. For example, a transgenic mouse can have ahuman light chain transgene and either a human heavy chain transgene orhuman heavy chain transchromosome, such that the mouse produces humananti-CLDN6 antibodies when immunized with CLDN6 antigen and/or cellsexpressing CLDN6. The human heavy chain transgene can be integrated intothe chromosomal DNA of the mouse, as is the case for transgenic mice,e.g., HuMAb mice, such as HCo7 or HCo12 mice, or the human heavy chaintransgene can be maintained extrachromosomally, as is the case fortranschromosomal (e.g., KM) mice as described in WO 02/43478. Suchtransgenic and transchromosomal mice may be capable of producingmultiple isotypes of human monoclonal antibodies to CLDN6 (e.g., IgG,IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.

According to the invention, the term “therapeutic effector moiety” meansany molecule which may exert a therapeutic effect. According to theinvention, a therapeutic effector molecule is preferably selectivelyguided to a cell which expresses CLDN6 and includes anticancer agents,radioisotopes, toxins, cytostatic or cytolytic drugs, etc. Anticanceragents comprise, for example, aminoglutethimide, azathioprine, bleomycinsulfate, busulfan, carmustine, chlorambucil, cisplatin,cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin,daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α,lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl,thioguanine, vinblastine sulfate and vincristine sulfate. Otheranticancer agents are described, for example, in Goodman and Gilman,“The Pharmacological Basis of Therapeutics”, 8th Edition, 1990,McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents (PaulCalabresi and Bruce A. Chabner). Toxins may be proteins such as pokeweedantiviral protein, cholera toxin, pertussis toxin, ricin, gelonin,abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxin residues mayalso be high energy-emitting radionuclides such as cobalt-60.

“Reduce” or “inhibit” as used herein means the ability to cause anoverall decrease, preferably of 5% or greater, 10% or greater, 20% orgreater, more preferably of 50% or greater, and most preferably of 75%or greater, in the level, e.g. in the level of proliferation of cells.The term “inhibit” or similar phrases includes a complete or essentiallycomplete inhibition, i.e. a reduction to zero or essentially to zero.

Terms such as “increase” or “enhance” preferably relate to an increaseor enhancement by about at least 10%, preferably at least 20%,preferably at least 30%, more preferably at least 40%, more preferablyat least 50%, even more preferably at least 80%, and most preferably atleast 100%.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect suchas induction of a humoral and/or cellular immune response, the strengthand/or duration of the induced immune reaction, or the specificity ofthe induced immune reaction. In the context of the present invention,the term “immunologically equivalent” is preferably used with respect tothe immunological effects or properties of a peptide or peptide variantused for immunization or an antibody. A particular immunologicalproperty is the ability to bind to antibodies and, where appropriate,generate an immune response, preferably by stimulating the generation ofantibodies. For example, an amino acid sequence is immunologicallyequivalent to a reference amino acid sequence if said amino acidsequence when exposed to the immune system of a subject induces animmune reaction, preferably antibodies, having a specificity of reactingwith the reference amino acid sequence, such as the reference amino acidsequence forming part of CLDN6.

The term “immune effector functions” in the context of the presentinvention includes any functions mediated by components of the immunesystem that result in the inhibition of tumor growth and/or inhibitionof tumor development, including inhibition of tumor dissemination andmetastasis. Preferably, immune effector functions result in killing oftumor cells. Preferably, the immune effector functions in the context ofthe present invention are antibody-mediated effector functions. Suchfunctions comprise complement dependent cytotoxicity (CDC),antibody-dependent cell-mediated cytotoxicity (ADCC), induction ofapoptosis in the cells carrying the tumor-associated antigen, e.g.CLDN6, for example, by binding of the antibody to a surface antigen,and/or inhibition of proliferation of the cells carrying thetumor-associated antigen, preferably ADCC and/or CDC. Thus, antibodiesthat are capable of mediating one or more immune effector functions arepreferably able to mediate killing of cells by inducing CDC-mediatedlysis, ADCC-mediated lysis, apoptosis, homotypic adhesion, and/orphagocytosis, preferably by inducing CDC-mediated lysis and/orADCC-mediated lysis. Antibodies may also exert an effect simply bybinding to tumor-associated antigens on the surface of a tumor cell. Forexample, antibodies may block the function of the tumor-associatedantigen or induce apoptosis just by binding to the tumor-associatedantigen on the surface of a tumor cell.

The antibodies, compositions and methods described herein can be used totreat a subject with a tumor disease, e.g., a disease characterized bythe presence of tumor cells expressing CLDN6. Examples of tumor diseaseswhich can be treated and/or prevented encompass all CLDN6 expressingcancers and tumor entities including those described herein.

The antibodies, compositions and methods described herein may also beused for immunization or vaccination to prevent a disease describedherein.

According to the invention, the term “disease” refers to anypathological state, including cancer, in particular those forms ofcancer described herein.

“Diseases involving cells expressing CLDN6” means according to theinvention that expression of CLDN6 in cells of a diseased tissue ororgan is preferably increased compared to the state in a healthy tissueor organ. An increase refers to an increase by at least 10%, inparticular at least 20%, at least 50%, at least 100%, at least 200%, atleast 500%, at least 1000%, at least 10000% or even more. In oneembodiment, expression is only found in a diseased tissue, whileexpression in a healthy tissue is repressed. According to the invention,diseases involving or being associated with cells expressing CLDN6include tumor diseases such as cancer diseases. Furthermore, accordingto the invention, tumor diseases such as cancer diseases preferably arethose wherein the tumor cells or cancer cells express CLDN6.

According to the invention, the term “tumor” or “tumor disease” refersto a swelling or lesion formed by an abnormal growth of cells (calledneoplastic cells or tumor cells). By “tumor cell” is meant an abnormalcell that grows by a rapid, uncontrolled cellular proliferation andcontinues to grow after the stimuli that initiated the new growth cease.Tumors show partial or complete lack of structural organization andfunctional coordination with the normal tissue, and usually form adistinct mass of tissue, which may be either benign, pre-malignant ormalignant.

A benign tumor is a tumor that lacks all three of the malignantproperties of a cancer. Thus, by definition, a benign tumor does notgrow in an unlimited, aggressive manner, does not invade surroundingtissues, and does not spread to non-adjacent tissues (metastasize).Common examples of benign tumors include moles and uterine fibroids.

The term “benign” implies a mild and nonprogressive disease, and indeed,many kinds of benign tumors are harmless to the health. However, someneoplasms which are defined as “benign tumors” because they lack theinvasive properties of a cancer, may still produce negative healtheffects. Examples of this include tumors which produce a “mass effect”(compression of vital organs such as blood vessels), or “functional”tumors of endocrine tissues, which may overproduce certain hormones(examples include thyroid adenomas, adrenocortical adenomas, andpituitary adenomas).

Benign tumors typically are surrounded by an outer surface that inhibitstheir ability to behave in a malignant manner. In some cases, certain“benign” tumors may later give rise to malignant cancers, which resultfrom additional genetic changes in a subpopulation of the tumor'sneoplastic cells. A prominent example of this phenomenon is the tubularadenoma, a common type of colon polyp which is an important precursor tocolon cancer. The cells in tubular adenomas, like most tumors whichfrequently progress to cancer, show certain abnormalities of cellmaturation and appearance collectively known as dysplasia. Thesecellular abnormalities are not seen in benign tumors that rarely ornever turn cancerous, but are seen in other pre-cancerous tissueabnormalities which do not form discrete masses, such as pre-cancerouslesions of the uterine cervix. Some authorities prefer to refer todysplastic tumors as “pre-malignant”, and reserve the term “benign” fortumors which rarely or never give rise to cancer.

Neoplasm is an abnormal mass of tissue as a result of neoplasia.Neoplasia (new growth in Greek) is the abnormal proliferation of cells.The growth of the cells exceeds, and is uncoordinated with that of thenormal tissues around it. The growth persists in the same excessivemanner even after cessation of the stimuli. It usually causes a lump ortumor. Neoplasms may be benign, pre-malignant or malignant.

“Growth of a tumor” or “tumor growth” according to the invention relatesto the tendency of a tumor to increase its size and/or to the tendencyof tumor cells to proliferate.

Preferably, a “tumor disease” according to the invention is a cancerdisease, i.e. a malignant disease, and a tumor cell is a cancer cell.Preferably, a “tumor disease” is characterized by cells expressing CLDN6and a tumor cell expresses CLDN6.

Cancer (medical term: malignant neoplasm) is a class of diseases inwhich a group of cells display uncontrolled growth (division beyond thenormal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis (spread to other locations in thebody via lymph or blood). These three malignant properties of cancersdifferentiate them from benign tumors, which are self-limited, and donot invade or metastasize. Most cancers form a tumor but some, likeleukemia, do not.

A cell expressing CLDN6 preferably is a tumor cell or cancer cell,preferably of the tumors and cancers described herein. Preferably, suchcell is a cell other than a placental cell.

Cancers are classified by the type of cell that resembles the tumor and,therefore, the tissue presumed to be the origin of the tumor. These arethe histology and the location, respectively.

The term “cancer” according to the invention comprises leukemias,seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas,rectal cancer, endometrial cancer, kidney cancer, adrenal cancer,thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervicalcancer, intestinal cancer, liver cancer, colon cancer, stomach cancer,intestine cancer, head and neck cancer, gastrointestinal cancer, lymphnode cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear,nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer ofthe uterus, ovarian cancer and lung cancer and the metastases thereof.Examples thereof are lung carcinomas, mamma carcinomas, prostatecarcinomas, colon carcinomas, renal cell carcinomas, cervicalcarcinomas, or metastases of the cancer types or tumors described above.The term cancer according to the invention also comprises cancermetastases.

Preferred tumor diseases or cancers according to the invention areselected from the group consisting of ovarian cancer, in particularovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer,including small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC), in particular squamous cell lung carcinoma and adenocarcinoma,gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skincancer, in particular basal cell carcinoma and squamous cell carcinoma,malignant melanoma, head and neck cancer, in particular malignantpleomorphic adenoma, sarcoma, in particular synovial sarcoma andcarcinosarcoma, bile duct cancer, cancer of the urinary bladder, inparticular transitional cell carcinoma and papillary carcinoma, kidneycancer, in particular renal cell carcinoma including clear cell renalcell carcinoma and papillary renal cell carcinoma, colon cancer, smallbowel cancer, including cancer of the ileum, in particular small boweladenocarcinoma and adenocarcinoma of the ileum, testicular embryonalcarcinoma, placental choriocarcinoma, cervical cancer, testicularcancer, in particular testicular seminoma, testicular teratoma andembryonic testicular cancer, and uterine cancer, and the metastaticforms thereof.

Particularly preferred tumor diseases or cancers according to theinvention are selected from the group consisting of ovarian cancer, lungcancer, metastatic ovarian cancer and metastatic lung cancer.Preferably, the ovarian cancer is an ovarian carcinoma or an ovarianadenocarcinoma. Preferably, the lung cancer is a carcinoma or anadenocarcinoma, and preferably is bronchiolar cancer such as abronchiolar carcinoma or bronchiolar adenocarcinoma. In one embodiment,the tumor cell is a cell of such a cancer. Metastatic ovarian cancersinclude metastatic ovarian carcinomas and metastatic ovarianadenocarcinomas, and metastatic lung cancers include metastatic lungcarcinomas, metastatic lung adenocarcinomas, metastatic bronchiolarcarcinomas, and metastatic bronchiolar adenocarcinomas.

The main types of lung cancer are small cell lung carcinoma (SCLC) andnon-small cell lung carcinoma (NSCLC). There are three main sub-types ofthe non-small cell lung carcinomas: squamous cell lung carcinoma,adenocarcinoma, and large cell lung carcinoma. Adenocarcinomas accountfor approximately 10% of lung cancers. This cancer usually is seenperipherally in the lungs, as opposed to small cell lung cancer andsquamous cell lung cancer, which both tend to be more centrally located.

Skin cancer is a malignant growth on the skin. The most common skincancers are basal cell cancer, squamous cell cancer, and melanoma.Malignant melanoma is a serious type of skin cancer. It is due touncontrolled growth of pigment cells, called melanocytes.

According to the invention, a “carcinoma” is a malignant tumor derivedfrom epithelial cells. This group represents the most common cancers,including the common forms of breast, prostate, lung and colon cancer.

“Bronchiolar carcinoma” is a carcinoma of the lung, thought to bederived from epithelium of terminal bronchioles, in which the neoplastictissue extends along the alveolar walls and grows in small masses withinthe alveoli. Mucin may be demonstrated in some of the cells and in thematerial in the alveoli, which also includes denuded cells.

“Adenocarcinoma” is a cancer that originates in glandular tissue. Thistissue is also part of a larger tissue category known as epithelialtissue. Epithelial tissue includes skin, glands and a variety of othertissue that lines the cavities and organs of the body. Epithelium isderived embryologically from ectoderm, endoderm and mesoderm. To beclassified as adenocarcinoma, the cells do not necessarily need to bepart of a gland, as long as they have secretory properties. This form ofcarcinoma can occur in some higher mammals, including humans. Welldifferentiated adenocarcinomas tend to resemble the glandular tissuethat they are derived from, while poorly differentiated may not. Bystaining the cells from a biopsy, a pathologist will determine whetherthe tumor is an adenocarcinoma or some other type of cancer.Adenocarcinomas can arise in many tissues of the body due to theubiquitous nature of glands within the body. While each gland may not besecreting the same substance, as long as there is an exocrine functionto the cell, it is considered glandular and its malignant form istherefore named adenocarcinoma. Malignant adenocarcinomas invade othertissues and often metastasize given enough time to do so. Ovarianadenocarcinoma is the most common type of ovarian carcinoma. It includesthe serous and mucinous adenocarcinomas, the clear cell adenocarcinomaand the endometrioid adenocarcinoma.

“Cystadenocarcinoma” is a malignant form of a surface epithelial-stromaltumor, a type of ovarian cancer.

Surface epithelial-stromal tumors are a class of ovarian neoplasms thatare thought to be derived from the ovarian surface epithelium (modifiedperitoneum) or from ectopic endometrial or Fallopian tube (tubal)tissue. This group of tumors accounts for the majority of all ovariantumors.

“Choriocarcinoma” is a malignant, trophoblastic and aggressive cancer,usually of the placenta. It is characterized by early hematogenousspread to the lungs.

Renal cell carcinoma also known as renal cell cancer or renal celladenocarcinoma is a kidney cancer that originates in the lining of theproximal convoluted tubule, the very small tubes in the kidney thatfilter the blood and remove waste products. Renal cell carcinoma is byfar the most common type of kidney cancer in adults and the most lethalof all the genitorurinary tumors. Distinct subtypes of renal cellcarcinoma are clear cell renal cell carcinoma and papillary renal cellcarcinoma. Clear cell renal cell carcinoma is the most common form ofrenal cell carcinoma. When seen under a microscope, the cells that makeup clear cell renal cell carcinoma appear very pale or clear. Papillaryrenal cell carcinoma is the second most common subtype. These cancersform little finger-like projections (called papillae) in some, if notmost, of the tumors.

A sarcoma is a malignant tumor derived from connective tissue, ormesenchymal cells. This is in contrast to carcinomas, which are ofepithelial origin. A synovial sarcoma is a rare form of cancer whichusually occurs near to the joints of the arm or leg. It is one of thesoft tissue sarcomas.

A germ cell tumor is a neoplasm derived from germ cells. Germ celltumors can be cancerous or non-cancerous tumors. Germ cells normallyoccur inside the gonads (ovary and testis). Germ cell tumors thatoriginate outside the gonads (e.g. in head, inside the mouth, neck,pelvis; in fetuses, babies, and young children most often found on thebody midline, particularly at the tip of the tailbone) may be birthdefects resulting from errors during development of the embryo.

The two major classes of germ cell tumors are the seminomas andnon-seminomas, wherein non-seminomas include: teratocarcinoma, embryonalcarcinoma, yolk sac tumors, choriocarcinoma and differentiated teratoma.Most cell lines from non-seminomas are equivalent to embryonalcarcinomas, that is, they are composed almost entirely of stem cellswhich do not differentiate under basal conditions, though some mayrespond to inducers of differentiation such as retinoic acid.

Teratocarcinoma refers to a germ cell tumor that is a mixture ofteratoma with embryonal carcinoma, or with choriocarcinoma, or withboth. This kind of mixed germ cell tumor may be known simply as ateratoma with elements of embryonal carcinoma or choriocarcinoma, orsimply by ignoring the teratoma component and referring only to itsmalignant component: embryonal carcinoma and/or choriocarcinoma.

Lymphoma and leukemia are malignancies derived from hematopoietic(blood-forming) cells.

Blastic tumor or blastoma is a tumor (usually malignant) which resemblesan immature or embryonic tissue. Many of these tumors are most common inchildren.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor, i.e. a secondary tumor ormetastatic tumor, at the target site depends on angiogenesis. Tumormetastasis often occurs even after the removal of the primary tumorbecause tumor cells or components may remain and develop metastaticpotential. In one embodiment, the term “metastasis” according to theinvention relates to “distant metastasis” which relates to a metastasiswhich is remote from the primary tumor and the regional lymph nodesystem.

The cells of a secondary or metastatic tumor are like those in theoriginal tumor. This means, for example, that, if ovarian cancermetastasizes to the liver, the secondary tumor is made up of abnormalovarian cells, not of abnormal liver cells. The tumor in the liver isthen called metastatic ovarian cancer, not liver cancer.

In ovarian cancer, metastasis can occur in the following ways: by directcontact or extension, it can invade nearby tissue or organs located nearor around the ovary, such as the fallopian tubes, uterus, bladder,rectum, etc.; by seeding or shedding into the abdominal cavity, which isthe most common way ovarian cancer spreads. Cancer cells break off thesurface of the ovarian mass and “drop” to other structures in theabdomen such as the liver, stomach, colon or diaphragm; by breakingloose from the ovarian mass, invading the lymphatic vessels and thentraveling to other areas of the body or distant organs such as the lungor liver; by breaking loose from the ovarian mass, invading the bloodsystem and traveling to other areas of the body or distant organs.

According to the invention, metastatic ovarian cancer includes cancer inthe fallopian tubes, cancer in organs of the abdomen such as cancer inthe bowel, cancer in the uterus, cancer in the bladder, cancer in therectum, cancer in the liver, cancer in the stomach, cancer in the colon,cancer in the diaphragm, cancer in the lungs, cancer in the lining ofthe abdomen or pelvis (peritoneum), and cancer in the brain. Similarly,metastatic lung cancer refers to cancer that has spread from the lungsto distant and/or several sites in the body and includes cancer in theliver, cancer in the adrenal glands, cancer in the bones, and cancer inthe brain.

A relapse or recurrence occurs when a person is affected again by acondition that affected them in the past. For example, if a patient hassuffered from a tumor disease, has received a successful treatment ofsaid disease and again develops said disease said newly developeddisease may be considered as relapse or recurrence. However, accordingto the invention, a relapse or recurrence of a tumor disease may butdoes not necessarily occur at the site of the original tumor disease.Thus, for example, if a patient has suffered from ovarian tumor and hasreceived a successful treatment a relapse or recurrence may be theoccurrence of an ovarian tumor or the occurrence of a tumor at a sitedifferent to ovary. A relapse or recurrence of a tumor also includessituations wherein a tumor occurs at a site different to the site of theoriginal tumor as well as at the site of the original tumor. Preferably,the original tumor for which the patient has received a treatment is aprimary tumor and the tumor at a site different to the site of theoriginal tumor is a secondary or metastatic tumor.

By “treat” is meant to administer a compound or composition as describedherein to a subject in order to: prevent or eliminate a tumor or reducethe size of a tumor or the number of tumors in a subject; arrest or slowthe growth of a tumor in a subject; inhibit or slow the development of anew tumor or tumor metastasis in a subject; decrease the frequency orseverity of symptoms and/or recurrences in a subject who currently hasor who previously has had cancer; and/or prolong, i.e. increase thelifespan of the subject.

In particular, the term “treatment of a disease” includes curing,shortening the duration, ameliorating, preventing, slowing down orinhibiting progression or worsening, or preventing or delaying the onsetof a disease or the symptoms thereof.

By “being at risk” is meant a subject, i.e. a patient, that isidentified as having a higher than normal chance of developing a tumordisease, in particular cancer, compared to the general population. Inaddition, a subject who has had, or who currently has, a tumor disease,in particular cancer is a subject who has an increased risk fordeveloping cancer, as such a subject may continue to develop cancer.Subjects who currently have, or who have had, a tumor also have anincreased risk for tumor metastases.

The term “immunotherapy” relates to a treatment involving a specificimmune reaction. In the context of the present invention, terms such as“protect”, “prevent”, “prophylactic”, “preventive”, or “protective”relate to the prevention or treatment or both of the occurrence and/orthe propagation of a tumor in an individual and, in particular, tominimizing the chance that a subject will develop a tumor or to delayingthe development of a tumor. For example, a person at risk for a tumor,as described above, would be a candidate for therapy to prevent a tumor.

A prophylactic administration of an immunotherapy, for example, aprophylactic administration of the composition of the invention,preferably protects the recipient from the development of tumor growth.A therapeutic administration of an immunotherapy, for example, atherapeutic administration of the composition of the invention, may leadto the inhibition of the progress/growth of the tumor. This comprisesthe deceleration of the progress/growth of the tumor, in particular adisruption of the progression of the tumor, which preferably leads toelimination of the tumor.

The term “in vivo” relates to the situation in a subject.

The terms “subject”, “individual” or “patient” are used interchangeablyand relate to vertebrates, preferably mammals. For example, mammals inthe context of the present invention are humans, non-human primates,domesticated animals such as dogs, cats, sheep, cattle, goats, pigs,horses etc., laboratory animals such as mice, rats, rabbits, guineapigs, etc. as well as animals in captivity such as animals of zoos. Theterm “animal” as used herein also includes humans. The term “subject”may also include a patient, i.e., an animal, preferably a human having adisease, preferably a disease associated with expression of CLDN6,preferably a tumor disease such as a cancer.

As part of the composition for an immunization or a vaccination,preferably one or more agents as described herein are administeredtogether with one or more adjuvants for inducing an immune response orfor increasing an immune response. The term “adjuvant” relates tocompounds which prolongs or enhances or accelerates an immune response.The composition of the present invention preferably exerts its effectwithout addition of adjuvants. Still, the composition of the presentapplication may contain any known adjuvant. Adjuvants comprise aheterogeneous group of compounds such as oil emulsions (e.g., Freund'sadjuvants), mineral compounds (such as alum), bacterial products (suchas Bordetella pertussis toxin), liposomes, and immune-stimulatingcomplexes. Examples for adjuvants are monophosphoryl-lipid-A (MPLSmithKline Beecham). Saponins such as QS21 (SmithKline Beecham), DQS21(SmithKline Beecham; WO 96/33739), QS7, QS17, QS18, and QS-L1 (So etal., 1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvants,complete Freund's adjuvants, vitamin E, montanid, alum, CpGoligonucleotides (Krieg et al., 1995, Nature 374: 546-549), and variouswater-in-oil emulsions which are prepared from biologically degradableoils such as squalene and/or tocopherol.

According to the invention, a “sample” may be any sample usefulaccording to the present invention, in particular a biological samplesuch a tissue sample, including bodily fluids, and/or a cellular sampleand may be obtained in the conventional manner such as by tissue biopsy,including punch biopsy, and by taking blood, bronchial aspirate, sputum,urine, feces or other body fluids. According to the invention, the term“sample” also includes processed samples such as fractions or isolatesof biological samples, e.g. nucleic acid and peptide/protein isolates.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (Hall (1995) Science 268:1432-1434), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds comprisecostimulating molecules provided in the form of proteins or nucleicacids such as B7-1 and B7-2 (CD80 and CD86, respectively).

The therapeutically active compounds of the invention may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitonealy, intramuscularly, subcutaneously ortransdermally. Preferably, antibodies are therapeutically administeredby way of a lung aerosol.

In a further embodiment, antibodies of the invention can be formulatedto prevent or reduce their transport across the placenta. This can bedone by methods known in the art, e.g., by PEGylation of the antibodiesor by use of F(ab)2′ fragments. Further references can be made to“Cunningham-Rundles C, Zhuo Z, Griffith B, Keenan J. (1992) Biologicalactivities of polyethylene-glycol immunoglobulin conjugates. Resistanceto enzymatic degradation. J. Immunol. Methods, 152: 177-190; and to“Landor M. (1995) Maternal-fetal transfer of immunoglobulins, Ann.Allergy Asthma Immunol. 74: 279-283.

The compositions of the invention are administered in effective amounts.An “effective amount” refers to the amount which achieves a desiredreaction or a desired effect alone or together with further doses. Inthe case of treatment of a particular disease or of a particularcondition, the desired reaction preferably relates to inhibition of thecourse of the disease. This comprises slowing down the progress of thedisease and, in particular, interrupting or reversing the progress ofthe disease. The desired reaction in a treatment of a disease or of acondition may also be delay of the onset or a prevention of the onset ofsaid disease or said condition.

An effective amount of a composition of the invention will depend on thecondition to be treated, the severeness of the disease, the individualparameters of the patient, including age, physiological condition, sizeand weight, the duration of treatment, the type of an accompanyingtherapy (if present), the specific route of administration and similarfactors. Accordingly, the doses of the compositions of the inventionadministered may depend on various of such parameters. In the case thata reaction in a patient is insufficient with an initial dose, higherdoses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

The pharmaceutical compositions of the invention are preferably sterileand contain an effective amount of the therapeutically active substanceto generate the desired reaction or the desired effect.

The pharmaceutical compositions of the invention are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible preparation. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers, supplementing immunity-enhancing substancessuch as adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines,saponin, GM-CSF and/or RNA and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the invention. Pharmacologicallyand pharmaceutically compatible salts of this kind comprise in anonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallycompatible salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier. The term “carrier” refers to anorganic or inorganic component, of a natural or synthetic nature, inwhich the active component is combined in order to facilitateapplication. According to the invention, the term “pharmaceuticallycompatible carrier” includes one or more compatible solid or liquidfillers, diluents or encapsulating substances, which are suitable foradministration to a patient. The components of the pharmaceuticalcomposition of the invention are usually such that no interaction occurswhich substantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the invention may contain suitablebuffer substances such as acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the invention may be in the form of capsules, tablets,lozenges, solutions, suspensions, syrups, elixirs or in the form of anemulsion, for example.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

EXAMPLES

The techniques and methods used herein are described herein or carriedout in a manner known per se and as described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methodsincluding the use of kits and reagents are carried out according to themanufacturers' information unless specifically indicated.

Example 1 Materials and Methods

A. Generation of Murine Antibodies Against CLDN6

a. Generation of Expression Vectors Encoding Full Length CLDN6 and CLDN6Fragments

A non-natural, codon-optimized DNA sequence (SEQ ID NO: 10) encodingfull length CLDN6 (SEQ ID NO: 2) was prepared by chemical synthesis(GENEART AG, Germany) and cloned into the pcDNA3.1/myc-His vector(Invitrogen, USA) yielding the vector p3953. Insertion of a stop codonallowed the expression of CLDN6 protein without being fused to thevector encoded myc-His tag. Expression of CLDN6 was tested by Westernblot, flow cytometry and immunofluorescence analyses using commerciallyavailable anti-CLDN6 antibodies (ARP, 01-8865; R&D Systems, MAB3656).

In addition, a codon-optimized DNA sequence (SEQ ID NO: 11) coding forthe putative extracellular domain 2 (EC2) fragment of CLDN6 (SEQ ID NO:5) as a fusion with an N-terminal Ig kappa leader derived signal peptidefollowed by 4 additional amino acids to ensure a correct signalpeptidase cleavage site (SEQ ID NO: 12) was prepared and cloned into thepcDNA3.1/myc-His vector yielding the vector p3974. Prior toimmunization, expression of the EC2 fragment was confirmed byimmunofluorescence microscopy on transiently transfected andparaformaldehyde (PFA)-fixed CHO-K1 cells using a commercially availableanti-myc antibody (Cell Signaling, MAB 2276).

b. Generation of Cell Lines Stably Expressing CLDN6

HEK293 and P3X63Ag8U.1 cell lines stably expressing CLDN6 were generatedby standard techniques using the vector p3953.

c. Immunizations

MuMAB 5F2D2: Balb/c mice were immunized with 25 μg of p3974 plasmid DNAtogether with 4 μl PEI-mannose (PEI-Man; in vivo-jetPEI™-Man fromPolyPlus Transfection) (150 mM PEI-Man in H₂O with 5% Glucose) byintraperitoneal injection on days 0, 16 and 36. On days 48 and 62 micewere immunized by intraperitoneal injection with 2×10⁷ P3X63Ag8U.1myeloma cells transfected with p3953 vector to stably express CLDN6. Thecells administered on day 62 had been irradiated with 3000 rad prior toinjection.

MuMAB 27A: Balb/c mice were immunized by intraperitonal injection with2×10⁷ P3X63Ag8U.1 myeloma cells transfected with p3953 vector to stablyexpress CLDN6 on day 0 and 13. Mice developing tumors were boosted byintraperitonal injection with 2×10⁷ HEK293 cells stably transfected withp3953 vector on day 21. After three days, mice were sacrificed andsplenocytes were prepared. 5×10⁷ splenocytes were transplanted intoanother Balb/c mouse by intravenous injection. On day 35, 49, 67 and 104the transplanted mice were immunized by intraperitoneal injection with2×10⁷ P3X63Ag8U.1 myeloma cells stably transfected with p3953 vectortogether with HPLC-purified phosphorothioate-modified CpGoligodeoxynucleotides (PTO-CpG-ODN) (50 μg; 5′-TCCATGACGTTCCTGACGTT;Eurofins MWG Operon, Germany). Prior administration, the cells weretreated with mitomycin-C (5 μg/ml, Sigma-Aldrich, M4287).

MuMAB 36A:C57BL/6 mice were immunized with 25 μg of p3974 plasmid DNAtogether with 4 μl PEI-mannose (PEI-Man; in vivo-jetPEI™-Man fromPolyPlus Transfection) (150 mM PEI-Man in H₂O with 5% Glucose) byintraperitoneal injection on days 0, 16 and 36. On days 55, 69 and 85mice were immunized by intraperitoneal injection with 2×10⁷ P3X63Ag8U.1myeloma cells transfected with p3953 vector to stably express CLDN6. Onday 85 cells were administered together with HPLC-purified PTO-CpG-ODN(50 μg in PBS; 5′-TCCATGACGTTCCTGACGTT; Eurofins MWG Operon, Germany).

The presence of antibodies directed against CLDN6 in sera of mice wasmonitored by immunofluorescence microscopy using CHO-K1 cellsco-transfected with nucleic acids encoding CLDN6 and GFP. To this end,24 h following transfection, PFA-fixed or non-fixed cells were incubatedwith a 1:100 dilution of sera from immunized mice for 45 min at roomtemperature (RT). Cells were washed, incubated with an Alexa555-labeledanti-mouse Ig antibody (Molecular Probes) and subjected to fluorescencemicroscopy.

For generation of monoclonal antibodies, mice with detectable anti-CLDN6immune responses were boosted four days prior to splenectomy byintraperitonal injection of 2×10⁷ HEK293 cells stably transfected withp3953 vector.

d. Generation of Hybridomas Producing Murine Monoclonal AntibodiesAgainst CLDN6

6×10⁷ splenocytes isolated from an immunized mouse were fused with 3×10⁷cells of the mouse myeloma cell line P3X63Ag8.653 (ATCC, CRL 1580) usingPEG 1500 (Roche, CRL 10783641001). Cells were seeded at approximately5×10⁴ cells per well in flat bottom microtiter plates and cultivated forabout two weeks in RPMI selective medium containing 10% heat inactivatedfetal bovine serum, 1% hybridoma fusion and cloning supplement (HFCS,Roche, CRL 11363735), 10 mM HEPES, 1 mM sodium pyruvate, 4.5% glucose,0.1 mM 2-mercaptoethanol, 1× penicillin/streptomycin and 1×HATsupplement (Invitrogen, CRL 21060). After 10 to 14 days, individualwells were screened by flow cytometry for anti-CLDN6 monoclonalantibodies. Antibody secreting hybridomas were subcloned by limitingdilution and again tested for anti-CLDN6 monoclonal antibodies. Thestable subclones were cultured to generate small amounts of antibody intissue culture medium for characterization. At least one clone from eachhybridoma which retained the reactivity of the parent cells (tested byflow cytometry) was selected. Nine-vial-cell banks were generated foreach clone and stored in liquid nitrogen.

B. Flow Cytometry

To test the binding of monoclonal antibodies to CLDN6 and other claudinsHEK293T cells were transiently transfected with the correspondingclaudin-coding plasmid and the expression was analyzed by flowcytometry. In order to differentiate between transfected andnon-transfected cells, HEK293T cells were co-transfected with afluorescence marker as a reporter. 24 h post transfection cells wereharvested with 0.05% Trypsin/EDTA, washed with FACS buffer (PBScontaining 2% FCS and 0.1% sodium azide) and resuspended in FACS bufferat a concentration of 2×10⁶ cells/ml. 100 μl of the cell suspension wereincubated with the appropriate antibody at indicated concentrations for30 min at 4° C. The commercially available mouse anti-claudin antibodiesanti-CLDN6 (R&D, CRL MAB3656), anti-CLDN3 (R&D, MAB4620) and anti-CLDN4(R&D, MAB4219) served as positive controls, whereas mouse IgG2b (Sigma,CRL M8894) served as isotype control. The cells were washed three timeswith FACS buffer and incubated with an allophycocyanin (APC)-conjugatedanti-mouse IgG 1+2a+2b+3a specific secondary antibody (Dianova, CRL115-135-164) for 30 min at 4° C. The cells were washed twice andresuspended in FACS buffer. The binding was analyzed by flow cytometryusing a BD FACSArray. The expression of the fluorescence marker wasplotted on the horizontal axis against the antibody binding on thevertical axis.

The binding of monoclonal antibodies to cell lines that endogenouslyexpress CLDN6 was analyzed in a similar manner.

C. Immunoblot Analysis

NEC8 cells were analyzed for CLDN6, 3, 4 and 9 expression by immunoblotanalysis. As positive control HEK293T cells were transiently transfectedwith either CLDN6, 3, 4, 9 or a mock plasmid as negative control. Cellswere harvested in Laemmli buffer, lysed and subjected to SDS-PAGE. Thegel was blotted and stained with an anti-CLDN3 (Invitrogen, 34-1700),anti-CLDN4 (Invitrogen, 32-9400), anti-CDLN6 (ARP, 01-8865) oranti-CLDN9 (Santa Cruz, sc-17672) antibody, respectively. Afterincubation with a peroxidase labelled secondary antibody the blot wasdeveloped with ECL reagent and visualized using a LAS-3000 imager(Fuji).

D. CDC Analysis

Complement dependent cytotoxicity (CDC) was determined by measuring thecontent of intracellular ATP in non-lysed cells after the addition ofhuman complement to the target cells incubated with anti-CLDN6antibodies. As a very sensitive analytical method the bioluminescentreaction of luciferase was used for measuring ATP.

CHO-K1 cells stably transfected with CLDN6 (CHO-K1-CLDN6) were harvestedwith 0.05% Trypsin/EDTA, washed twice with X-Vivo 15 medium (Lonza,BE04-418Q) and suspended at a concentration of 1×10⁷ cells/ml in X-Vivo15 medium. 250 μl of the cell suspension were transferred into a 0.4 cmelectroporation cuvette and mixed with 7 μg of in vitro transcribed RNAencoding for luciferase (luciferase IVT RNA). The cells wereelectroporated at 200 V and 300 μF using a Gene Pulser Xcell (Bio Rad).After electroporation, the cells were suspended in 2.4 ml pre-warmedD-MEM/F12 (1:1) with GlutaMax-I medium (Invitrogen, 31331-093)containing 10% (v/v) FCS, 1% (v/v) penicillin/streptomycin and 1.5 mg/mlG418. 50 μl of the cell suspension per well were seeded into a white96-well PP-plate and incubated at 37° C. and 7.5% CO₂. 24 h postelectroporation 50 μl monoclonal murine anti-CLDN6 antibodies in 60%RPMI (containing 20 mM HEPES) and 40% human serum (serum pool obtainedfrom six healthy donors) were added to the cells at indicatedconcentrations. 10 μl 8% (v/v) Triton X-100 in PBS per well were addedto total lysis controls, whereas 10 μl PBS per well were added to maxviable cells controls and to the actual samples. After an incubation of80 min at 37° C. and 7.5% CO₂ 50 μl luciferin mix (3.84 mg/mlD-luciferin, 0,64 U/ml ATPase and 160 mM HEPES in ddH₂O) were added perwell. The plate was incubated in the dark for 45 min at RT. Thebioluminescence was measured using a luminometer (Infinite M200, TECAN).Results are given as integrated digital relative light units (RLU).

The specific lysis is calculated as follows:

${{specific}\mspace{14mu}{{lysis}\lbrack\%\rbrack}} = {100 - \left\lbrack {\frac{\left( {{sample} - {{total}\mspace{14mu}{lysis}}} \right)}{\left( {\max\left\lbrack {{viable}\mspace{14mu}{cells}\mspace{31mu}{total}\mspace{14mu}{lysis}} \right)} \right\rbrack} \times 100} \right\rbrack}$

-   -   max viable cells: 10 μl PBS, without antibody    -   total lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without        antibody        E. ADCC Analysis

Chimerized monoclonal anti-CLDN6 antibodies were analysed for theircapability to induce antibody-dependent cellular cytotoxicity (ADCC)against endogenously CLDN6 expressing NEC8 cells and NEC8 cells withCLDN6 knock-down (NEC8 LVTS2 54) in a luciferase-based assay system.

NEC8 or NEC8 LVTS2 54 target cells were harvested with 0.05%Trypsin/EDTA, washed twice with X-Vivo 15 medium and 2.5×10⁶ cells wereelectroporated with 7 μg luciferase IVT RNA (200 V, 400 μF) using a GenePulser Xcell (Bio Rad). After electroporation, the cells were suspendedin 2.4 ml pre-warmed RPMI containing 10% FCS. 50 μl of the cellsuspension (5×10⁴ cells) per well were seeded into a white 96-wellPP-plate and incubated at 37° C., 5% CO₂ for 6 h. Human peripheral bloodmononuclear cells (PBMCs) were enriched from the blood of healthy donorsusing Ficoll (Ficoll-Paque™ Plus, GE Healthcare, 17-1440-03). The PBMCswere suspended in X-Vivo 15 (Lonza, BE04-418Q) supplemented with 5%heat-inactivated human serum and incubated at 37° C. and 5% CO₂. Afterincubation for 2-4 h the supernatant was enriched in natural killer (NK)cells. 25 μl antibodies diluted in PBS at indicated concentrations wereadded to NEC8 cells. Enriched NK cells were added at a ratio of 20:1(effector to target cells) and the samples were incubated for 24 h at37° C. and 5% CO₂. Lysis of cells was determined by measuring thecontent of intracellular ATP with luciferase as described in “CDC”.

F. Early Treatment Assay

For early antibody treatments 5×10⁶ HEK293-CLDN6 cells (HEK293 cellsstably expressing CLDN6) in 200 μl PBS were subcutaneously inoculatedinto the flank of athymic Nude-Foxn1^(nu) mice. HEK293-mock cells wereused as negative controls. Each experimental group consisted of eight6-8 week-old female mice. (In case of mice engrafted with HEK293-CLDN6or -mock cells, respectively, the saline control groups consisted of tenmice.) Three days after inoculation 200 μg of purified murine monoclonalantibodies muMAB 5F2D2, 27A and 36A were applied for 46 days byalternating intravenous and intraperitoneal injections twice a week.Experimental groups treated with PBS served as a negative controls. Thetumor volume (TV=(length×width²)/2) was monitored bi-weekly. TV isexpressed in mm³, allowing construction of tumor growth curves overtime. When the tumor reached a volume greater than 1500 mm³ mice werekilled.

Example 2 Binding of Antibodies Obtained According to the Invention toClaudins

The binding of the murine monoclonlal antibodies muMAB 5F2D2, 27A and36A to human CLDN6, 3, 4 and 9 was analyzed by flow cytometry usingHEK293T cells transiently expressing the corresponding human claudin.HEK293T were co-transfected with a fluorescence marker to distinguishbetween non-transfected (Q3 population) and transfected (Q4 population)cells. The antibody concentration used was the concentration thatsaturated binding to CLDN6 (25 μg/ml). The expression of human CLDN6, 3,4 and 9 was confirmed with commercially available monoclonal antibodiesagainst human Claudin-6 (R&D Systems, MAB3656), human Claudin-3 (R&DSystems, MAB4620) and human Claudin-4 (R&D Systems, MAB 4219).

MuMAB 5F2D2, 27A and 36A antibodies showed strong binding to cellsexpressing CLDN6, while they did not bind to cells expressing CLDN3 orCLDN4; see FIG. 1.

MuMAB 5F2D2, 27A and 36A antibodies were incubated at differentconcentrations (0.01, 0.1, 1, 10, 100, 1000, 10000 and 25000 ng/ml) withHEK293T cells transiently expressing human CLDN6, CLDN3, CLDN4 or CLDN9.Binding was detected by flow cytometry; see FIGS. 2, 3 and 4. The y-axisrepresents the percentage of cells bound by antibody (Q2 population)while the x-axis represents the concentration of antibody used.

MuMAB 5F2D2 showed strong binding to human CLDN6 and weak binding tohuman CLDN9. MuMAB 27A showed strong binding to human CLDN6 and veryweak binding to human CLDN9. MuMAB 36A showed strong binding to humanCLDN6 and virtually no binding to human CLDN9. None of the antibodiesinteracted with either human CLDN3 or 4.

For determining relative affinities, the binding of anti-CLDN6antibodies to human CLDN6 stably expressed on the surface of HEK293cells was analysed by flow cytometry. In the saturation bindingexperiment the concentration of the antibodies was plotted against theFACS signals (median of fluorescence intensity); see FIG. 5. The EC50(antibody concentration that binds to half the binding sites atequilibrium) was calculated by nonlinear regression. The results showthat the antibodies have characteristic binding patterns. MuMAB 5F2D2and 27A exhibited low EC50 values (EC50 350-450 ng/ml) and saturation ofbinding was achieved at low concentrations whereas muMAB 36A did notshow saturation of binding even at the highest concentration.

Example 3 Effector Functions of Antibodies Obtained According to theInvention

The CDC activity of anti-CLDN6 antibodies was analysed using aluciferase-dependent assay to detect endogenous ATP within non-lysedcells. To this end, CHO-K1 cells stably expressing human CLDN6 weretreated with different concentrations of MuMAB 5F2D2, 27A or 36A oranti-CLDN6 (R&D Systems, MAB3656) as an internal control.

MuMAB 5F2D2 showed CDC activity in a dose-dependent manner; see FIG. 6.MuMAB 27A exhibited dose-dependent CDC activity whereas muMAB 36A wasnot able to induce CDC in vitro; see FIG. 7.

The ability of the chimeric anti-CLDN6 antibody chimAB 5F2D2 to induceantibody-dependent cell-mediated cytotoxicity (ADCC) on endogenouslyCLDN6 expressing NEC8 and NEC8 LVTS2 54 (CLDN6 knock-down) cells wasdetermined; see FIG. 8. The chimeric anti-CLDN6 antibody chimAB 5F2D2and the positive control Herceptin induced ADCC on NEC8 cells witheffector cells of two different donors in a dose dependent manner. Theefficiency to induce ADCC on NEC8 LVTS2 54 cells (CLDN6 knock-down) wasstrongly decreased with chimAB 5F2D2 compared to NEC8 parental cells,proving the target specificity of chimAB 5F2D2.

Example 4 Therapeutic Efficacy of Antibodies Obtained According to theInvention

The therapeutic effect of muMAB 5F2D2 was tested in an early treatmentxenograft model wherein stably transfected HEK293-CLDN6 and HEK293-mockxenografts were engrafted into athymic Nude-Foxn1^(nu) mice.

MuMAB 5F2D2 showed specific and strong tumor growth inhibition in miceengrafted with HEK293 cells stably expressing human CLDN6; see FIG. 9.MuMAB 5F2D2 had no effect on the tumor growth in mice engrafted withmock control cells. Separate saline control groups of mice received PBSas vehicle at injection volumes equivalent to the applied antibodies.

In addition, a Kruskal-Wallis test showed that tumor volumes weresignificantly reduced at day 28 (and thereafter) after treatment withmuMAB 5F2D2; see FIG. 10. Furthermore, mice treated with the monoclonalmurine anti-CLDN6 antibody muMAB 5F2D2 showed prolonged survivalcompared to PBS control groups. The antibody-dependent effect was notobserved in mice engrafted with HEK293-mock cells as a control group;see FIG. 11.

Similarly, testing of muMAB 27A and muMAB 36A in an early treatmentxenograft model showed specific and strong tumor growth inhibition inmice engrafted with HEK293 cells stably expressing human CLDN6; seeFIGS. 12 and 13. Furthermore, muMAB 27A and 36A were effective inprolonging the survival of engrafted mice; see FIG. 14.

Example 5 CLDN6 as a Cancer Target in Germ Cell Tumors

CLDN3, 4, 6 and 9 expression was tested by immunoblot analysis in NEC8cells. The testicular germ cell tumor cell line NEC8 only showedexpression of CLDN6 (left panel) but not of CLDN3, 4 or 9, respectively(right panels); see FIG. 15. The specificities of the anti-CLDN3, 4 and9 antibodies used were tested by Western blot using HEK293T cellstransiently transfected with expression vectors encoding for thecorresponding human claudin.

CLDN6 surface expression on NEC8 cells was analyzed using flowcytometry. The commercially available anti-CLDN6 antibody (R&D Systems,MAB3656) detected expression of CLDN6 on NEC8 cells; see FIG. 16. MouseIgG2b (Sigma, CRL M8894) was used as an isotype control.

The therapeutic effect of muMAB 5F2D2 in an early treatment xenograftmodel using mice engrafted with the tumor cell line NEC8 was tested.Compared to the saline control group muMAB 5F2D2 showed specific andstrong tumor growth inhibition in mice engrafted with NEC8 cells thatendogenously express human CLDN6; see FIG. 17. The Kruskal-Wallis testshows that tumor volumes were reduced at day 21 and 42 after treatmentwith muMAB 5F2D2; see FIG. 18.

The invention claimed is:
 1. An anti-claudin 6 (CLDN6) antibody selectedfrom the group consisting of (i) an antibody produced by a hybridomadeposited under the accession no. DSM ACC3059 (GT512muMAB 36A), DSMACC3058 (GT512muMAB 27A), or DSM ACC3057 (GT512muMAB 5F2D2), (ii) ahumanized or chimerized form of said antibody, (iii) an antigen bindingfragment of said antibody, and (iv) a synthetic form of said antibody;wherein the antibody or fragment of (i), (ii), (iii), and (iv) iscapable of binding to CLDN6 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 6, or having the amino acid sequence encoded by SEQ IDNO: 1; and wherein the antibody or fragment of (i), (ii), (iii), and(iv), without an attached therapeutic effector moiety, when administeredto a patient having a tumor characterized by tumor cells expressingCLDN6 inhibits growth of the tumor by binding to CLDN6.
 2. The antibodyof claim 1, wherein the antibody is specific for CLDN6.
 3. Apharmaceutical composition comprising the antibody of claim 1 and apharmaceutically acceptable carrier.
 4. The antibody of claim 1, whereinthe tumor is selected from the group consisting of ovarian cancer,ovarian adenocarcinoma, ovarian teratocarcinoma, lung cancer, small celllung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous celllung carcinoma, adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, basal cell carcinoma, squamouscell carcinoma, malignant melanoma, head and neck cancer, malignantpleomorphic adenoma, sarcoma, synovial sarcoma, carcinosarcoma, bileduct cancer, cancer of the urinary bladder, transitional cell carcinoma,papillary carcinoma, kidney cancer, renal cell carcinoma, clear cellrenal cell carcinoma, papillary renal cell carcinoma, colon cancer,small bowel cancer, cancer of the ileum, small bowel adenocarcinoma,adenocarcinoma of the ileum, testicular embryonal carcinoma, placentalchoriocarcinoma, cervical cancer, testicular cancer, testicularseminoma, testicular teratoma, embryonic testicular cancer, uterinecancer, a germ cell tumor disease, a teratocarcinoma, an embryonalcarcinoma, and the metastatic forms thereof.
 5. The antibody of claim 1,wherein the tumor is a germ cell tumor of the testis.
 6. The antibody ofclaim 1, wherein the antibody or fragment of (i), (ii), (iii), and (iv)binds to an epitope within SEQ ID NO: 3 or SEQ ID NO:
 5. 7. An antibodywhich binds to the same epitope as the antibody of claim
 1. 8. Apharmaceutical composition comprising the antibody of claim 6 and apharmaceutically acceptable carrier.
 9. An anti-claudin 6 antibodyselected from the group consisting of: (i) an antibody produced by ahybridoma deposited under the accession no. DSM ACC3059 (GT512muMAB36A), DSM ACC3058 (GT512muMAB 27A), or DSM ACC3057 (GT512muMAB 5F2D2);(ii) a humanized or chimerized form of the antibody under (i); and (iii)an antigen binding fragment of the antibody under (i) or (ii).
 10. Theantibody of claim 9, wherein the antibody is a monoclonal antibody. 11.The antibody of claim 9, wherein the antibody is a chimerized form ofthe antibody produced by a hybridoma deposited under the accession no.DSM ACC3059 (GT512muMAB 36A).
 12. The antibody of claim 9, wherein theantibody is a chimerized form of the antibody produced by a hybridomadeposited under the accession no. DSM ACC3058 (GT512muMAB 27A).
 13. Theantibody of claim 9, wherein the antibody is a chimerized form of theantibody produced by a hybridoma deposited under the accession no. DSMACC3057 (GT512muMAB 5F2D2).
 14. The antibody of claim 9, wherein theantibody is a humanized form of the antibody produced by a hybridomadeposited under the accession no. DSM ACC3059 (GT512muMAB 36A).
 15. Theantibody of claim 9, wherein the antibody is a humanized form of theantibody produced by a hybridoma deposited under the accession no. DSMACC3058 (GT512muMAB 27A).
 16. The antibody of claim 9, wherein theantibody is a humanized form of the antibody produced by a hybridomadeposited under the accession no. DSM ACC3057 (GT512muMAB 5F2D2). 17.The antibody of claim 9, wherein the antibody is attached to at leastone therapeutic effector moiety.
 18. The antibody of claim 17, whereinthe therapeutic effector moiety is a radiolabel, cytotoxin or cytotoxicenzyme.
 19. A pharmaceutical composition comprising the anti-claudin 6antibody of claim 9 and a pharmaceutically acceptable carrier.